Inhibitors of notch signalling pathway and use thereof in treatment of cancers

ABSTRACT

The present invention relates to use of inhibitors of Notch signalling pathway selected from the group consisting of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), its derivatives, in treating and/or preventing cancers.

FIELD OF THE INVENTION

The present invention relates to the use of inhibitors of Notchsignalling pathway in particular 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) (CAS number 218457-67-1) and its derivatives, in the treatmentand/or prevention of cancers.

BACKGROUND OF THE INVENTION

The Notch signalling pathway represents a critical component in themolecular circuits that control cell fate during development, cellsurvival and cell proliferation (Shih IeM, Wang T L in Cancer Res 2007;67(5):1879-82). Aberrant activation of this pathway contributes totumorigenesis. The Notch family members are being revealed as oncogenesin an ever-increasing number of cancers. The role of Notch in humancancer has been highlighted recently by the presence of activatingmutations and amplification of Notch genes in human cancer and by thedemonstration that genes in the Notch signalling pathway could bepotential therapeutic targets. It has become clear that one of the majortherapeutic targets in the Notch pathway are the Notch receptors, inwhich γ-secretase inhibitors prevent the generation of the oncogenic(intracellular) domain of Notch molecules and suppress the Notchactivity.

Though significant progress has been made in dissecting the complexworkings of this signalling pathway, there are very limited optionsavailable for Notch inhibitors. However, the pioneering class of Notchinhibitors is already in clinical trials for few cancer types, such asγ-secretase inhibitors MK0752 of Merck Sharp & Dohme Corp. MK0752, andRO4929097 (Roche), a synthetic small molecule, inhibits the Notchsignalling pathway, which may result in induction of growth arrest andapoptosis in tumor cells in which the Notch signalling pathway isoveractivated.

One of the drawbacks of use of γ-secretase inhibitors to block Notchsignaling, as currently on the market or under investigation, is theirwide range of additional targets such as amyloid precursor protein aswell as non-selectivity in blocking Notch signalling via all fourligands (Notch1, 2, 3 and 4). Due to their ability to block Notchsignalling via all four receptors γ-secretase inhibitors are known tocause goblet cell metaplasia in the intestine. In addition, some of thehematological malignancies and solid tumors harbor mutations in theNotch receptors (such as chromosomal translocations) resulting inconstitutive expression of dominant active form of NICD independent ofcleavage by γ-secretase complex. Therefore these tumors fail to respondto γ-secretase inhibitors treatment.

Therefore, there is still a need to identify and develop furtherspecific and selective inhibitors of Notch signalling pathway useful fortreating and/or preventing cancers.

SUMMARY OF THE INVENTION

The present invention concerns an 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) of Formula I

or one of its derivatives having Notch signalling pathway inhibitionproperties, salts, solvates, tautomers, isomers thereof for use in thetreatment and/or prevention of a cancer.

A further object of the present invention is to provide a pharmaceuticalcomposition comprising pharmaceutical composition comprising6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I, or one of itsderivatives having Notch signalling pathway inhibition properties, orpharmaceutically acceptable salts, solvates, tautomers, isomers thereof,and a pharmaceutically acceptable carrier.

The invention also contemplates a kit comprising one or more doses of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), or one of its derivativeshaving Notch signalling pathway inhibition properties, for use in amethod for treatment and/or prevention of cancer, optionally withreagents and/or instructions for use.

A further object of the invention is to provide the use of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I or one of itsderivatives having Notch signalling pathway inhibition properties, forinhibiting in vitro or in vitro the Notch signalling pathway in cells.

Another object of the invention is to provide a method of treating asubject for Notch dependent cancer.

DESCRIPTION OF THE FIGURES

FIG. 1 shows 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) (CAS number218457-67-1) blocks NICD mediated Notch signalling activation. A)N1-HeLa cells were co-transfected with pcDNA3.Notch1 expression plasmid,pGL4.26-12×CSL luciferase and SV40 renilla plasmids. DL4- and N1-HeLacells were cocultured in a 96 well plate in 1:1 ratio (20,000:20,000cells/well) and treated with DMSO or with 2, 5 and 10 μM of I3 and DAPTfor 24 hours. The Notch pathway activation was measured by quantifyingNotch signalling driven luciferase reporter assay. Treatment of DL4:N1coculture assay with 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) andDAPT causes a concentration dependent decrease in Notch signallingactivation. B) HeLa cells were transfected with NICD and treated withDMSO or with 2, 5, 10, 20 and 40 μM of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3). As a control co-culturedcells were also treated with 5, 10, 20 and 40 μM of DAPT. The pathwayactivation was measured using Notch driven luciferase reporter assay.6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment of NICD expressingcells led to an attenuation of the signalling, while DAPT treatment hadno effect on Notch signalling activation mediated by NICD. C) DL4:N1 andDL4:N2 coculture assay was treated with I3 and DAPT (each 10 μM) for 24hours. The effect of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) andDAPT on DL4-N1 and DL4-N2 driven pathway activation was measured byNotch driven luciferase activity. Both I3 and DAPT treatment blockNotch1 and Notch2 induced pathway activation. D)6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) inhibits pathway activationvia intracellular domains of Notch1 (NICD) and Notch2 (N2-ICD).

FIG. 2 shows 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) mediatedinhibition of Notch signalling can be rescued with increasingconcentration of MAML1. HeLa cells were co-transfected 800 ng of NICD+3μg of pCDNA3.1 or 800 ng of NICD+1 μg of MAML1-FLAG or 800 ng of NICD+3μg of MAML1-FLAG expression vectors. To measure Notch pathwayactivation, pGL4.26-12×CSL luciferase plasmid was also introduced intothe cells. SV40 renilla was used as an internal control. Cellstransfected with different combinations and amounts of plasmid weretreated with DMSO or increasing concentration of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) (1, 2.5, 5 and 10 μM) for 24hours. 12×CSL driven luciferase activity was measured using dualluciferase assay system. In the absence of MAML1,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) could block Notch signallingactivations, but Notch inhibitory effect of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) was diminished withincreasing amount of MAML1.

FIG. 3 shows 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) inhibits Notchsignalling and downregulates its target genes in human cancer celllines. A) RPMI 8402 cells were treated with DMSO,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) and DAPT (10 μM) for 24hours and analyzed for the expression of Notch target genes, Hes1, cMycand Dtx1 by qRT-PCR. Data normalized to HPRT as a house-keeping gene. B)Whole cell lysate from 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3)treated cells was analyzed by Western blot. Using antibodies againstNICD (Val1744), Hes1 and cMyc, the protein levels of NICD and Notchtarget genes were determined. C and D) The human T-ALL cell lines HPBALL and KOPTK1 were treated with DMSO or6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) for 24 hours. Western blotanalyses were performed using NICD (Val1744) and Hes1 specificantibodies. Tubulin served as a loading control. E) Whole cell lysatefrom DMSO, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treated PANC1cells (pancreatic cancer cell line) were analyzed by Western blot. Hes1protein levels were determined using Hes1 specific antibodies.Statistical analyses were done using student's two-tailed t.test. *=pvalue<0.05.

FIG. 4 shows 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) induces aproliferative block in human cancer cells. Human T-ALL cell lines RPMI8402 and KOPTK1, and pancreatic cancer cell line PANC1 as well as nRasdriven melanoma cells were seeded in a 96 well plate and treated with 10μM concentration of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) and DAPTfor several days. Their growth inhibitory effects were compared withcells treated with equal amount of DMSO. Using Alamar blue assay, thegrowth kinetics of RPMI 8402 and KOPTK1 were followed for up to 6 days,while PANC1 and nRas melanoma cells were monitored for 4 days.6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment of RPMI 8402,KOPTK1. PANC1 and nRas melanoma cells caused a significant reduction intheir growth potential. Statistical analyses were done using student'st.test. *=p value<0.05. ns=not significant.

FIG. 5 shows 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) blocks NICDdependent growth of human cancer cells. A) DND41-Parental and DND41-NICDcells were treated with 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) andDAPT for 24 hours. Western blot analyses were carried out for Hes1protein using Hes1 specific antibodies. Both DAPT and6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) caused a downregulation ofHes1 in DND41-Parental cells. DND41-NICD cells showed a downregulationof Hes1 only when treated with 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3). B) Five thousand DND41-Parental cells were seeded and treated withDMSO, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) and DAPT in a 96 wellplate. Growth kinetics of the parental cell line was followed over 5days using Alamar blue readout. Treatment of DND41-Parental cell linewith both 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) and DAPT caused aproliferation arrest. C) Similarly, DND41-NICD cells treated with DMSO,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) and DAPT and their growthkinetics were monitored using Alamar blue readout over 5 days. Thetreatment of DND41-NICD cells with DAPT did not have a significantimpact on their proliferation, while6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment induced aproliferation arrest. D) Human breast cancer cell line HCC1187 harbors aSEC22B-Notch2 chromosomal translocation, thus leading to an expressionof constitutively active form of NICD independent of cleavage by theγ-secretase complex. This mutation renders this cell line insensitive toγ-secretase inhibitor treatment. E) Two thousand HCC1187 cells wereseeded per well in a 96 well plate. The cells were treated with DMSO,γ-secretase inhibitor DAPT and I3 for 6 days. Alamar blue readout wastaken at day 0, day 2, day 4 and day 6. Eight replicates were used foreach treatment and time point. The treatment of HCC1187 human breastcancer cell line with γ-secretase inhibitor DAPT did not alter thegrowth kinetics when compared to DMSO treated counterparts, while I3treatment caused statistically significant inhibition of cellproliferation. P values were calculated using Student's t.test. *=pvalue<0.05. ns=not significant.

FIG. 6 shows 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) induces G0/G1cell cycle arrest and apoptosis in human T cell acute lymphoblasticleukemia cell lines and human breast cancer cell line HCC1187. A) Humanleukemic cell lines (RPMI8402, CUTL1, KOPTK1, TALL1 and HPBALL) weretreated with I3 (10 μM). Percentage of Annexin V positive (apoptotic)cell population was measured using flow cytometry. B) Cell cycleanalyses: RPMI8402, KOPTK1 and TALL1 cell lines were treated with I3 (10μM) and stained with Ki67 and Hoechst stain to determine cell cyclestatus. The cell cycle analyses suggest that I3 treatment causes 20-30%increase in cells arrested in G0/G1 phase of the cell cycle. C) HCC1187cells were treated with DMSO or 10 μM of I3 and percentage of apoptoticpopulation was measured using Annexin V stain. D) I3 treated HCC1187cells analyzed for cell cycle status. Ki67 and Hoechst stain revealedthat I3 induces G0/G1 arrest in HCC1187 cells.

FIG. 7 shows 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) mimics geneticloss of Notch2 signalling phenotype in the spleen. Loss of Notchsignalling in the spleen leads to a reduction in Marginal Zone B cells(MZB) cells in the spleen. A) Schematics of the experimental plan. B)Mice (n=2) were treated with oil or 25 mg/kg of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) for 7 consecutive days.Spleens were analyzed on day 8. Using B220 specific antibodies, B cellsin the spleen were identified. MZB cells within the B cell compartmentwere detected using antibodies against CD23 and CD21 cell surfacemarkers. The treatment of mice with6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) causes a significantreduction in the percentage of MZB cells in the spleen. B)6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment causes a reductionin the absolute numbers of MZB cells in the spleen when compared tovehicle treated animals.

FIG. 8 shows 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatmentincreases latency of leukemia development in mice. A) NOD/SCID γc^(−/−)mice were injected with 1×10⁶ HPB ALL (luciferase expressing) cells. Onday 15, leukemic cells were established in the bone marrow. Mice weretreated with oil or 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) forevery day. Mice were imaged on day 27 using Caliper IVIS (Xenogen) liveimaging system. Red and blue colour indicates the intensity ofluciferase signal and correlates with the number of leukemic cells. B)6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment blocks RPMI 8402leukemic cell growth in xenotransplantation assay. NOD/SCID γc^(−/−)mice were transplanted with 5×10⁵ RPMI 8402 (luciferase expressing)cells. Leukemia development was followed using Caliper IVIS (Xenogen)live imaging system. On day 13, a daily treatment was started using oil(n=3) or 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) (n=4). Animals weretreated for 27 days (end point of the experiment).

FIG. 9 shows 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatmentblocks MMTV-ErbB2 mouse mammary tumors. A) Hes1 protein expressionlevels in MMTV-ErbB2 mammary tumors and normal age matched mammaryglands (M.G) were compared using Anti-Hes1 antibody. Western blotanalyses showed a very high expression of Hes1 protein in MMTV-ErbB2mammary tumors. Tubulin served as a loading control. B) A single cellsuspension of MMTV-ErbB2 mammary tumor was prepared and 1×10⁶ cells wereinjected into the cleared fat pad of recipient FVB mice. Tumor formationwas monitored on a regular basis. Once the tumor developed to a volumeof 100-300 mm³, mice were treated with oil (n=2) or 25 mg/kg of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) (n=2). Tumor volume wasmeasured every 6-7 days. Mice treated with6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) exhibited a slow tumorprogression compared to oil treated mice.

FIG. 10 shows Notch inhibitory activity of chemical derivatives of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3). Different chemicalderivatives of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) were testedin DL4-N1 coculture assay and Notch activity levels were measured usingNotch driven luciferase reporter gene. Derivatives I3-A, I3-B, I3-C,I3-E, I3-G, I3-H, I3-M and I3-N exhibit anti-Notch activity comparableto 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), while derivatives I3-Fand I3-I appear to have enhanced activity.

DETAILED DESCRIPTION OF THE INVENTION

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. The publications andapplications discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention. Inaddition, the materials, methods, and examples are illustrative only andare not intended to be limiting.

In the case of conflict, the present specification, includingdefinitions, will control. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as is commonlyunderstood by one of skill in art to which the subject matter hereinbelongs. As used herein, the following definitions are supplied in orderto facilitate the understanding of the present invention.

As used herein, the term “comprise/comprising” is generally used in thesense of include/including, that is to say permitting the presence ofone or more features or components. The terms “comprise” and“comprising” also encompass the more restricted ones “consist” and“consisting”.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise.

For the ease of reading, the term “compound(s) of the invention” or“compound(s) according to the invention” used throughout the descriptionrefers to the compound 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) (CASnumber 218457-67-1), derivatives of said I3, salts or solvates of thecompound I3 or of the derivatives, and to isomers, includingenantiomers, stereoisomers, rotamers, tautomers and racemates of thecompound I3, chemical modified I3 compounds and derivatives of said I3compounds.

As used herein the terms “subject” is well-recognized in the art, and,refers to a mammal, including dog, cat, rat, mouse, monkey, cow, horse,goat, sheep, pig, camel, and, most preferably, a human. In someembodiments, the subject is a subject in need of treatment or a subjectwith a disease or disorder, such as cancer. However, in otherembodiments, the subject can be a normal subject or a subject who hasalready undergone a treatment against cancer. The term does not denote aparticular age or sex. Thus, adult, children and newborn subjects,whether male or female, are intended to be covered.

The terms “cancer”, “cancer cells”, “cell proliferative diseases” and“cell proliferative disorders” as used herein refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. According to the present invention, cancerrefers preferably to solid tumors, such as brain, breast, prostate,colorectum, kidney, lung, sarcoma, or melanoma and liquid tumors,affecting the blood, such as leukemia. More preferably according to thepresent invention, cancers are Notch dependent cancers selected from thegroup comprising T cell-Acute lymphoblastic leukemia (T-ALL), chronicmyeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Mantle celllymphoma, breast cancer, pancreatic cancer, prostate cancer, melanoma,brain tumors, tumor angiogenesis, colorectal cancer. Alternatively, theNotch dependent cancer is resistant to γ-secretase inhibitor treatment.Examples of γ-secretase inhibitor treatment comprise 1) Gamma secretaseinhibitor RO4929097 and Cediranib Maleate in treating patients withadvanced solid tumors (NCT01131234), 2) Gamma-Secretase InhibitorRO4929097 in Treating Young Patients With Relapsed or Refractory SolidTumors, CNS Tumors, Lymphoma, or T-Cell Leukemia (NCT01088763), 3) Studyof MK-0752 in combination with Tamoxifen or Letrozole to treat earlystage breast cancer (NCT00756717), 4) GDC-0449 and RO4929097 in treatingpatients with Advances or metastatic sarcoma (NCT01154452) 5) RO4929097and Erlotinib Hydrochloride in treating patients with stage IV orrecurrent Non-Small Cell Lung Cancer (NCT01193881), 6) Bicalutamide andRO4929097 in treating patients with previously treated prostate cancer(NCT01200810), 7) RO4929097 in treating patients with recurrent invasiveGliomas (NCT01269411), 8) A Notch signaling pathway inhibitor forpatients with T-cell Acute Lymphoblastic Leukemia/Lymphoma (ALL)(NCT00100152) and 9) RO4929097 in treating patients with metastaticcolorectal cancer (NCT01116687).

The Notch signalling pathway is evolutionarily conserved and the basicmolecular players in this pathway are ligands (Delta and Jagged), Notchreceptors, and the transcription factors (Shih IeM, Wang T L in CancerRes 2007; 67(5):1879-82). Notch is a transmembrane heterodimericreceptor and there are four distinct members (Notch1, Notch2, Notch3 andNotch4) in humans and rodents. In a physiologic condition, binding ofthe Notch ligand to its receptor initiates Notch signalling by releasingthe intracellular domain of the Notch receptor (Notch-ICD) through acascade of proteolytic cleavages by both α-secretase (also called tumornecrosis factor-α-converting enzyme) and γ-secretase. The releasedintracellular Notch-ICD then translocates into the nucleus where itmodulates gene expression primarily by binding to a ubiquitoustranscription factor, CBF1, suppressor of hairless, Lag-1 (CSL). Thisbinding recruits transcription activators to the CSL complex andconverts it from a transcriptional repressor into an activator, whichturns on several downstream effectors. The physiologic functions ofNotch signalling are multifaceted, including maintenance of stem cells,specification of cell fate, and regulation of differentiation indevelopment as well as in oncogenesis.

In cancers, molecular genetic alterations, such as chromosomaltranslocation, point mutations, and chromosomal amplification at theNotch receptor loci, are the known mechanisms for constitutiveactivation of Notch pathway. Despite the different mechanisms, they allresult in increased levels of intracellular Notch-IC. The oncogenicpotential of Notch was first discovered in human T-cell acutelymphoblastic leukemia (T-ALL). While Notch1 signalling is essential fornormal development of T-cell progenitors, constitutive activation ofNotch1 signalling due to molecular genetic alterations is associatedwith T-ALL. For example, interstitial deletions of the extracellularportion of human Notch1 due to (7; 9) chromosomal translocation areassociated with ˜1% of T-ALL cases and activating point mutations ofNotch1 are present in about 50% of T-ALL cases. Formation of T-cellleukemia/lymphoma was observed in a Notch-ICD transgenic mouse model,which indicates a causal role of Notch activation in T-ALL development.In non-small cell lung cancer, chromosomal translocation (15; 19) hasbeen identified in a subset of tumors, and the translocation is thoughtto elevate Notch3 transcription in tumors. In ovarian cancer, Notch3gene amplification was found to occur in about 19% of tumors, andoverexpression of Notch3 was found in more than half of the ovarianserous carcinomas. Similarly, Notch signalling activation has been shownin the development of breast cancer. In animal models, constitutivelyactive Notch4 expression causes mammary tumors in mice andNotch1-activating mutations contribute to the development of T-ALL. Arecent study further shows that overexpression of activated Notch1 andNotch3 in transgenic mice blocks mammary gland development and inducesmouse breast tumors. Notch signalling activation has also beenimplicated in lung and bone metastasis of breast cancer cells.Overexpression of Notch3 is sufficient to induce choroid plexus tumorformation in a mouse model, suggesting a role of Notch3 in thedevelopment of certain types of brain tumors.

With the aim of conducting a High-Through put Screening (HTS) toidentify novel modulators (inhibitors) of Notch signalling, Applicantshave established a coculture assay to induce a ligand-receptor mediatedactivation of the pathway. The coculture assay was established usingNotch ligand DL4 and Notch1 receptor specifically, because DL4-N1ligand-receptor mediated pathway activation plays an important role inpathophysiological conditions such as tumor angiogenesis and the role ofNotch1 receptor in inducing T cell leukemia. Since this assay depends onthe expression and interaction between DL4 ligand and Notch1 receptor,it provides an opportunity to interrogate ligand-receptorinteractions-induced Notch signalling in a controlled manner. Theminiaturization of this assay into a 96 well plate and 384 well plateformat helped Applicants to adapt this assay to conduct HTS. The use ofthis coculture assay to screen siRNA or small molecule libraries canlead to the identification of proteins or chemical compounds that areable to modulate Notch signalling at different steps along the pathway.For example, a HTS using siRNA or small molecule libraries can yieldmodulators of the pathway able to act in the signal sending or signalreceiving cells. Small molecule or protein mediated alterations in therecycling or trafficking of the ligands and receptors to the plasmamembrane can potentially block the Notch pathway and could be studiedusing this assay. In addition, this assay can also help identifyproteins or chemical entities able to block ligand-receptorinteractions, ADAM10/17 mediated S2 cleavage or γ-secretase catalyzed S3cleavage of the Notch receptor, nuclear translocation of the active formof Notch or entities able to block transcriptional activation complex.

Applicants have also been able to screen three different chemicalcompound libraries (Microsource NIMDS, Prestwick and Maybridge Hitfinder) that have led to the identification of several chemicals, whichare able to block Notch signalling at different levels along thepathway.

The use of the Notch-independent renilla system as an internal controlallowed Applicants to eliminate cytotoxic chemical compounds, therebylimiting the rate of false positive hits. In addition, this cell-basedassay also helped to circumvent issues related to the cell permeabilityof the chemical compounds for further hit validation.

The development of DL4:N1 coculture assay system laid the foundation fora HTS campaign. This assay provided a robust and sensitive readoutsystem to identify novel modulators (inhibitors) of the Notch pathway.

Applicants identified several chemical compounds for their ability toblock the Notch pathway activation. Among those, they identified thecompound 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) (CAS number218457-67-1) for its ability to block the Notch pathway activation.

Thus, the present invention relates to6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I

for use in the treatment and/or prevention of a cancer.

The present invention also encompasses chemical modifications of the6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) (CAS number: 218457-67-1) toprolong their circulating lifetimes. Non-limiting examples of methodsfor transiently, or reversibly, pegylating drugs, includingpolypeptide-based drugs, are provided in U.S. Pat. No. 4,935,465 (issuedin Jun. 19, 1990) and U.S. Pat. No. 6,342,244 (issued Jan. 29, 2002);and in U.S. published applications number US2006/0074024. One skilled inthe art would typically find more details about PEG-based reagents in,for example, published applications WO2005047366, US2005171328, andthose listed on the NEKTAR PEG Reagent Catalog® 2005-2006 (NektarTherapeutics, San Carlos, Calif.).

The present invention further encompasses chemical derivatives of saidI3 having Notch signalling pathway inhibition properties. Applicantshave shown that 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) and itsderivatives target Notch signalling at the transcriptional activationcomplex in the nucleus, human tumors resistant to γ-secretase inhibitorsdue to above mentioned mutations are expected to respond to6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment. In addition,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) appears to selectivelytarget Notch signalling, thus limiting its off-target toxic effects.

These derivatives all share the follow common structure:

Preferably, in said derivatives X is O and position 3 (or para) is NH2.

Most preferably, the derivative having Notch signalling pathwayinhibition properties is selected from the non-limiting group comprising

wherein m is an integer selected from 1 to 4;

-   W is selected from H and halogens; the halogen is selected from F—,    Cl—, Br— or I—;-   R1, R2, R3, R4 are each independently selected from the group    consisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or    3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,    benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the    subscript n is an integer independently selected from 1 to 15;-   X is O, S, CR5R6, NR7, NHCOR8, or NHSO2R9; where R5, R6, R7, R8, R9    are each independently selected from the group consisting in    selected from the group consisting in H, phenyl, 2-, 3- or    4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,    thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,    or (CH2)_(n)CH3, the subscript n is an integer independently    selected from 1 to 15;-   Y is N or CH;-   Z is H, NO₂, OH, NR10R11 where R10 and R11 are each independently    selected from the group consisting in H and (CH₂)_(n)CH₃, NHCOR12    where R12 is selected from the group consisting of (CH₂)_(n)CH₃,    aromatic and heteroaromatics such as phenyl, naphthyl, pyrrolyl,    furanyl, thiofuranyl, pyrimidinyl, imidazolyl, COOR13 where R13 is    selected from the group consisting of H, (CH₂)_(n)CH₃, aromatic and    heteroaromatics such as phenyl, naphthyl, pyrrolyl, furanyl,    thiofuranyl, pyrimidinyl, imidazolyl, NHSO₂R14 where R14 is selected    from the group consisting of phenyl, 2-, 3- or 4, substituted    phenyl, naphthyl, heteroaromatics such as pyrrolyl, furanyl,    thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH₂)_(n)CH₃; the    subscript n is an integer independently selected from 1 to 15;

wherein m is an integer selected from 1 to 4;

-   W is selected from H and halogens; the halogen is selected from F—,    Cl—, Br— or I—;-   R4, R15 are each independently selected from the group consisting in    H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl,    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n    is an integer independently selected from 1 to 15;-   X is O, S, CR5R6, NR7, NHCOR8 or NHSO₂R9; R5, R6 and R7 are each    independently selected from the group consisting in H, phenyl, 2-,    3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,    thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,    (CH₂)_(n)CH₃; R8 and R9 are each independently selected from the    group consisting in phenyl, 2-, 3- or 4-substituted phenyl, 2- or    3-naphthyl, or heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 1 to 15;-   Y is N or CH;-   Z is H, NO₂, OH, NR10R11 where R¹⁰ and R¹¹ are each independently    selected from the group consisting in H, (CH₂)_(n)CH₃, NHCOR¹² where    R12 is (CH₂)_(n)CH₃, aromatic and heteroaromatics selected from the    group comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,    pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH₂)_(n)CH₃,    aromatic and heteroaromatics selected from the group comprising    phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,    imidazolyl, NHSO₂R14 where R14 is phenyl, 2-, 3- or 4, substituted    phenyl, naphthyl, heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃, the subscript n is an integer independently selected    from 1 to 15;

wherein m is an integer selected from 1 to 4;

-   W is selected from H and halogens; the halogen is selected from F—,    Cl—, Br— or I—;-   R4 is H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl,    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n    is an integer independently selected from 1 to 15;-   X is O, S, CR5R6, NR7, NHCOR8 or NHSO₂R9; R5, R6 and R7 are each    independently selected from the group consisting in H, phenyl, 2-,    3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,    thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,    (CH₂)_(n)CH₃; R8 and R9 are each independently selected from the    group consisting in phenyl, 2-, 3- or 4-substituted phenyl, 2- or    3-naphthyl, or heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 1 to 15;-   Y is N or CH;-   Z is H, NO₂, OH, NR10R11 where R¹⁰ and R¹¹ are each independently    selected from the group consisting in H, (CH₂)_(n)CH₃, NHCOR¹² where    R12 is (CH₂)_(n)CH₃, aromatic and heteroaromatics selected from the    group comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,    pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH₂)_(n)CH₃,    aromatic and heteroaromatics selected from the group comprising    phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,    imidazolyl, NHSO₂R14 where R14 is phenyl, 2-, 3- or 4, substituted    phenyl, naphthyl, heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃, the subscript n is an integer independently selected    from 1 to 15;

wherein m is an integer selected from 1 to 4;

-   W is selected from H and halogens; the halogen is selected from F—,    Cl—, Br— or I—;-   R1, R2, R3, R4 are each independently selected from the group    consisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or    3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,    benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the    subscript n is an integer independently selected from 1 to 15;-   X is O, S, CR5R6, NR7, NHCOR8 or NHSO₂R9; R5, R6 and R7 are each    independently selected from the group consisting in H, phenyl, 2-,    3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,    thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,    (CH₂)_(n)CH₃; R8 and R9 are each independently selected from the    group consisting in phenyl, 2-, 3- or 4-substituted phenyl, 2- or    3-naphthyl, or heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 1 to 15;-   Z is H, NO₂, OH, NR10R11 where R10 and R11 are each independently    selected from the group consisting in H, (CH₂)_(n)CH₃, NHCOR12 where    R12 is (CH₂)_(n)CH₃, aromatic and heteroaromatics selected form the    group comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,    pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH₂)_(n)CH₃,    aromatic and heteroaromatics selected from the group comprising    phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,    imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4, substituted    phenyl, naphthyl, heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 1 to 15;

wherein m is an integer selected from 1 to 4;

-   W is selected from H and halogens; the halogen is selected from F—,    Cl—, Br— or I—;-   R4, R15 are each independently selected from the group consisting in    H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl,    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n    is an integer independently selected from 0 to 15;-   X is O, S, CR5R6, NR7, NHCOR8 or NHSO₂R9; R5, R6 and R7 are each    independently selected from the group consisting in H, phenyl, 2-,    3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,    thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,    (CH₂)_(n)CH₃; R8 and R9 are each independently selected from the    group consisting in phenyl, 2-, 3- or 4-substituted phenyl, 2- or    3-naphthyl, or heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 0 to 15;-   Z is H, NO₂, OH, NR10R11 where R10 and R11 are each independently    selected from the group consisting in H, (CH₂)_(n)CH₃, NHCOR12 where    R12 is (CH₂)_(n)CH₃, aromatic and heteroaromatics selected form the    group comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,    pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH₂)_(n)CH₃,    aromatic and heteroaromatics selected from the group comprising    phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,    imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4, substituted    phenyl, naphthyl, heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 0 to 15;

wherein m is an integer selected from 1 to 3;

-   W is selected from H and halogens; the halogen is selected from F—,    Cl—, Br— or I—;-   R4 is H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl,    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n    is an integer independently selected from 0 to 15;-   X is O, S, CR5R6, NR7, NHCOR8 or NHSO₂R9; R5, R6 and R7 are each    independently selected from the group consisting in H, phenyl, 2-,    3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,    thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,    (CH₂)_(n)CH₃; R8 and R9 are each independently selected from the    group consisting in phenyl, 2-, 3- or 4-substituted phenyl, 2- or    3-naphthyl, or heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 0 to 15;-   Z is H, NO₂, OH, NR10R11 where R10 and R11 are each independently    selected from the group consisting in H, (CH₂)_(n)CH₃, NHCOR12 where    R12 is (CH₂)_(n)CH₃, aromatic and heteroaromatics selected form the    group comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,    pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH₂)_(n)CH₃,    aromatic and heteroaromatics selected from the group comprising    phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,    imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4, substituted    phenyl, naphthyl, heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 0 to 15;

wherein the heteroaromatic is an aminopyrrole, aminofurane,aminothiofurane, or a pyrimidine;

-   R1, R2, R3, R4 are each independently selected from the group    consisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or    3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,    benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the    subscript n is an integer independently selected from 0 to 15;-   X is O, S, CR5R6, NR7, NHCOR8, or NHSO2R9; where R5, R6, R7, R8, R9    are each independently selected from the group consisting in H,    phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl,    furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl,    tertButyl, or (CH2)_(n)CH3, the subscript n is an integer    independently selected from 0 to 15;-   Z is H, NO₂, OH, NR10R11 where R10 and R11 are each independently    selected from the group consisting in H, (CH₂)_(n)CH₃, NHCOR12 where    R12 is (CH₂)_(n)CH₃, aromatic and heteroaromatics selected form the    group comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,    pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH₂)_(n)CH₃,    aromatic and heteroaromatics selected from the group comprising    phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,    imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4, substituted    phenyl, naphthyl, heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 0 to 15;

wherein the subscript n is an integer independently selected from 1 to15;

-   the heteroaromatic is an aminopyrrole, aminofurane, aminothiofurane,    or a pyrimidine;-   R4, R15 are each independently selected from the group consisting in    H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl,    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n    is an integer independently selected from 0 to 15;-   X is O, S, CR5R6, NR7, NHCOR8 or NHSO₂R9; R5, R6 and R7 are each    independently selected from the group consisting in H, phenyl, 2-,    3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,    thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,    (CH₂)_(n)CH₃; R8 and R9 are each independently selected from the    group consisting in phenyl, 2-, 3- or 4-substituted phenyl, 2- or    3-naphthyl, or heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 0 to 15;-   Z is H, NO₂, OH, NR10R11 where R10 and R11 are each independently    selected from the group consisting in H, (CH₂)_(n)CH₃, NHCOR12 where    R12 is (CH₂)_(n)CH₃, aromatic and heteroaromatics selected form the    group comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,    pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH₂)_(n)CH₃,    aromatic and heteroaromatics selected from the group comprising    phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,    imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4, substituted    phenyl, naphthyl, heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 0 to 15;

wherein the heteroaromatic is an aminopyrrole, aminofurane,aminothiofurane, or a pyrimidine;

-   R4 is H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl,    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n    is an integer independently selected from 0 to 15;-   X is O, S, CR5R6, NR7, NHCOR8 or NHSO₂R9; R5, R6 and R7 are each    independently selected from the group consisting in H, phenyl, 2-,    3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,    thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,    (CH₂)_(n)CH₃; R8 and R9 are each independently selected from the    group consisting in phenyl, 2-, 3- or 4-substituted phenyl, 2- or    3-naphthyl, or heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 0 to 15;-   Z is H, NO₂, OH, NR10R11 where R10 and R11 are each independently    selected from the group consisting in H, (CH₂)_(n)CH₃, NHCOR12 where    R12 is (CH₂)_(n)CH₃, aromatic and heteroaromatics selected form the    group comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,    pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH₂)_(n)CH₃,    aromatic and heteroaromatics selected from the group comprising    phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,    imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4, substituted    phenyl, naphthyl, heteroaromatics selected from the group comprising    pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl,    (CH₂)_(n)CH₃; the subscript n is an integer independently selected    from 0 to 15;

Even more preferably, the derivative having Notch signalling pathwayinhibition properties is selected from the group consisting in

The invention also relates to salts or solvates of the compound I3,chemical modified I3 compounds and derivatives of said I3 compounds ofthe invention. Preferably, these salts and/or solvates arepharmaceutically acceptable. According to the present invention,pharmaceutically acceptable salts are produced from acidic inorganic ororganic compounds, or alkaline inorganic or organic compounds. As usedherein, the phrase “pharmaceutically acceptable salt” refers to a saltthat retains the biological effectiveness of the free acids and bases ofa specified compound and that is not biologically or otherwiseundesirable.

Unless specified otherwise, it is further understood that all isomers,including enantiomers, stereoisomers, rotamers, tautomers and racematesof the compound I3, chemical modified I3 compounds and derivatives ofsaid I3 compounds of the invention are contemplated as being part ofthis invention. The invention includes stereoisomers in optically pureform and in admixture, including racemic mixtures. Isomers can beprepared using conventional techniques, either by reacting opticallypure or optically enriched starting materials or by separating isomersof compounds of the present invention.

“Racemates” refers to a mixture of enantiomers.

“Stereoisomer” or “stereoisomers” refer to compounds that differ in thechirality of one or more stereocentres. Stereoisomers includeenantiomers and diastereomers. The compound I3, chemical modified I3compounds and derivatives of said I3 compounds of this invention mayexist in stereoisomeric form if they possess one or more asymmetriccentres or a double bond with asymmetric substitution and, therefore,can be produced as individual stereoisomers or as mixtures. Unlessotherwise indicated, the description is intended to include individualstereoisomers as well as mixtures. The methods for the determination ofstereochemistry and the separation of stereoisomers are well-known inthe art (see discussion in Chapter 4 of Advanced Organic Chemistry, 4thed., J. March, John Wiley and Sons, New York, 1992).

“Tautomer” refers to alternate forms of a compound that differ in theposition of a proton, such as enol-keto and imine-enamine tautomers, orthe tautomeric forms of heteroaryl groups containing a ring atomattached to both a ring —NH— moiety and a ring ═N-moiety such aspyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles.

A skilled person will know that, if compound I3, chemical modified I3compounds and derivatives of said I3 compounds of the invention containcharged group, a suitable counterion will be derived from an organic orinorganic acid. Such counterions include halide (such as chloride,bromide, fluoride, iodide), sulfate, phosphate, acetate, succinate,citrate, lactate, maleate, fumarate, palmitate, cholate, glutamate,glutarate, tartrate, stearate, salicylate, methanesulfonate,benzenesulfonate, sorbate, picrate, benzoate, cinnamate, and the like.If the polar moiety is a negatively charged group, a suitable counterionwill be selected from sodium, ammonium, barium, calcium, copper, iron,lithium, potassium and zinc, and the like.

Surprisingly, the chemical compound6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) was identified as apotential Notch inhibitor. Interestingly,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) was found to block NICDmediated pathway activation (FIG. 1). Because of its ability toattenuate NICD mediated Notch activation,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) is able to blockproliferation of NICD overexpressing leukemic cell lines which areresistant to DAPT (a-γ-secretase inhibitor,N-[N-(3,5-difluorophenacetyl-Lalanyl)]-(S)-phenylglycine t-butyl ester)(FIG. 5). The Notch inhibitory potential of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) was further confirmed by thedownregulation of Notch target genes in human T-ALL cell lines (FIG. 3)and Affymetrix geneChip array (data not shown). The fact that6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) can induce differentiationof C2C12 cells into MHC expressing multinucleated myotubes furthervalidated the anti-Notch role of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) (data not shown). The Notch pathway inhibition caused by6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) can be rescued by theoverexpression of MAML1 above certain levels. For example,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) was able to block thesignalling with 800 ng of NICD and 1 μg of MAML1 was transientlyintroduced into the cells, however when the amount of MAML1 wasincreased to 3 μg, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) was nolonger able to block the pathway activation (FIG. 2). These data suggestthat, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) may interfere with theNotch transcriptional activation complex thereby inhibiting thesignalling activation. Microscopic studies by introduction of MAML atlevels where 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) could stillblock the pathway activation showed that6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment does not impedeco-localization of NICD, MAML1 and CSL/RBP-jk in the sub-nuclearcompartments (data not shown). Without being bound to theory, one of thepossible mechanisms of action of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) could be to disrupt the recruitment of transcriptional coactivatorsto the core CSL/RBP-jk-NICD-MAML1 complex. Therefore, the status ofadditional coactivators involved in the formation of functionaltranscriptional activation complex still needs to be determined. Underphysiological conditions, following the formation ofCSL/RBP-jk-NICD-MAML1 complex, CBP/p300 histone acetyltransferase (HAT)is recruited to the complex leading to its autoacetylation andacetylation of histone 3 and 4.

6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), as well as derivativesthereof, were further investigated in an in vivo context to determinetheir Notch inhibitory as well as toxic side effects in the mice. Notchsignalling is essential for the maintenance of normal homoeostasis inthe intestine. Genetic ablation or pharmacological inhibition of Notch1and Notch2 signalling in the intestine leads to goblet cell metaplasiain the intestine. Since, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) hasbeen observed to block Notch1 and Notch2 mediated signalling, micetreated with 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) were expectedto develop goblet cell metaplasia. Surprisingly, treatment of mice with25 mg/kg of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) for 7 days (morethan a month in case of xenotransplants) did not perturb intestinalhomeostasis and without any indication of goblet cell accumulation (datanot shown). This unexpected outcome could be due to two reasons. Onepossible explanation could be that the concentration of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) (25 mg/kg) used is notsufficient to block Notch pathway activation in the intestine. However,a second more plausible explanation could be the differences in thecomposition of transcriptional activation complexes downstream of Notch1and Notch2 signalling. Due to these possible differences, Notch1 andNotch2 mediated signalling may have different sensitivities against6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment.

Notch signalling plays an important role in the regulation ofhematopoietic system. For instance, DL4-Notch1 signalling is essentialfor T cell development in the thymus. Notch2 and MAML1 mediated pathwayactivation is critical for Marginal Zone B (MZB) cells development inthe spleen. To address whether 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) can impair Notch dependent MZB cell development in the spleen,C57B16 mice were treated with 25 mg/kg of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) for 7 days and analyzed onday 8. Flow cytometry analyses using antibodies against B220, CD21 andCD23 revealed that 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatmentcauses a reduction the percentage and absolute numbers of MZB cells inthe spleen (FIG. 7).

Anti-cancer activity of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) wasinvestigated in transplant models for human diseases, namely T-cellleukemia and breast cancer. In these studies,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) has demonstrated aremarkable ability to slow down the progression and metastasis of veryaggressive form of leukemic cell lines (FIG. 8). In addition, in apreliminary study using breast cancer as a model of solid tumors,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment has led to a blockin tumor progression in the mice (FIG. 9).

The chemical compound 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) hasshown ability to block NICD mediated signalling. Therefore this compoundis useful in cancers where Notch driven tumors are resistant toγ-secretase inhibitor treatment.

The present invention also provides a pharmaceutical compositioncomprising 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of formula I, orone of its derivatives having Notch signalling pathway inhibitionproperties as described herein, or pharmaceutically acceptable salts,solvates, tautomers, isomers thereof, and a pharmaceutically acceptablecarrier. As to the appropriate carriers, reference may be made to thestandard literature describing these, e.g. to chapter 25.2 of Vol. 5 of“Comprehensive Medicinal Chemistry”, Pergamon Press 1990, and to“Lexikon der Hilfsstoffe für Pharmazie, Kosmetik and angrenzendeGebiete”, by H. P. Fiedler, Editio Cantor, 2002. The term“pharmaceutically acceptable carrier” means a carrier or excipient thatis useful in preparing a pharmaceutical composition that is generallysafe, and possesses acceptable toxicities. Acceptable carriers includethose that are acceptable for veterinary use as well as humanpharmaceutical use. A “pharmaceutically acceptable carrier” as used inthe specification and claims includes both one and more than one suchcarrier. Optionally, the pharmaceutical composition of the presentinvention further comprises one or more additional active agentsselected among the non limiting group comprising chemotherapeutic agentsfor treating cancer. Such chemotherapeutic agents may be selected amongthe group comprising, for example, Altretamine, Bleomycin, Busulphan,Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin,Cladribine, Crisantaspase, Cyclophosphamid, Cytarabine, Dacarbazine,Daunorubicin, Doxorubicin, Epirubicin, Etoposide, Fludarabine,Fluorouracil, Gemcitabine, Idarubicin, Ifosfamide, Irinotecan,Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin,Mitoxantrone, Oxaliplatin, Pentostatin, Procarbazine, Streptozocin,Taco, Temozolomide, Tioguanine/Thioguanine, Thiotepa, Topotecan,Treosulfan, Vinblastine, Vincristine, Vindesine and Vinorelbine.

The compounds of the invention, namely the6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) and derivatives thereof,that are used in the treatment and/or prevention of cancers can beincorporated into a variety of formulations and medicaments fortherapeutic administration. More particularly, one or more compound(s)as provided herein can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers, andcan be formulated into preparations in solid, semi-solid, liquid orgaseous forms, such as tablets, capsules, pills, powders, granules,dragees, gels, slurries, ointments, solutions, suppositories,injections, inhalants and aerosols. As such, administration of thecompounds can be achieved in various ways, including oral, buccal,rectal, parenteral, intraperitoneal, intradermal, transdermal,intracranial and/or intratracheal administration. Moreover, the compoundcan be administered in a local rather than systemic manner, in a depotor sustained release formulation. The compounds can be formulated withcommon excipients, diluents or carriers, and compressed into tablets, orformulated as elixirs or solutions for convenient oral administration,or administered by the intramuscular or intravenous routes. Thecompounds can be administered transdermally, and can be formulated assustained release dosage forms and the like. The compounds can beadministered alone, in combination with each other, or they can be usedin combination with other known compounds. Suitable formulations for usein the present invention are found in Remington's PharmaceuticalSciences (Mack Publishing Company (1985) Philadelphia, Pa., 17th ed.),which is incorporated herein by reference. Moreover, for a brief reviewof methods for drug delivery, see, Langer, Science (1990) 249:1527-1533,which is incorporated herein by reference.

The amount of a compound as provided herein that can be combined with acarrier material to produce a single dosage form will vary dependingupon the disease treated, the subject in need thereof, and theparticular mode of administration. However, as a general guide, suitableunit doses for the compounds of the present invention can, for example,preferably contain between 0.1 mg to about 1000 mg, between 1 mg toabout 500 mg, and between 1 mg to about 300 mg of the active compound.In another example, the unit dose is between 1 mg to about 100 mg. Suchunit doses can be administered more than once a day, for example, 2, 3,4, 5 or 6 times a day, but preferably 1 or 2 times per day, so that thetotal dosage for a 70 kg human adult is in the range of 0.001 to about15 mg per kg weight of subject per administration. A preferred dosage is0.01 to about 1.5 mg per kg weight of subject per administration, andsuch therapy can extend for a number of weeks or months, and in somecases, years. It will be understood, however, that the specific doselevel for any particular patient will depend on a variety of factorsincluding the activity of the specific compound employed; the age, bodyweight, general health, sex and diet of the individual being treated;the time and route of administration; the rate of excretion; other drugsthat have previously been administered; and the severity of theparticular disease undergoing therapy, as is well understood by those ofskill in the area. A typical dosage can be one 1 mg to about 100 mgtablet or 1 mg to about 300 mg taken once a day, or, multiple times perday, or one time-release capsule or tablet taken once a day andcontaining a proportionally higher content of active ingredient. Thetime-release effect can be obtained by capsule materials that dissolveat different pH values, by capsules that release slowly by osmoticpressure, or by any other known means of controlled release. It can benecessary to use dosages outside these ranges in some cases as will beapparent to those skilled in the art.

The present invention further provides a compound of the invention foruse in treating and/or preventing cancers.

As used herein, cancers are preferably Notch dependent cancers and areselected from the non limiting group comprising T cell-Acutelymphoblastic leukemia (T-ALL), chronic myeloid leukemia (CML), chroniclymphocytic leukemia (CLL), Mantle cell lymphoma, breast cancer,pancreatic cancer, prostate cancer, melanoma, brain tumors, tumorangiogenesis, and colorectal cancer.

Preferably, the compounds of the present invention(6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), its derivatives) can bealso used in the treatment of cancers where Notch dependent cancers areresistant to γ-secretase inhibitor treatment. Notch signalling dependenthuman tumors resistant to γ-secretase inhibitor treatment can bedetermined by the levels of NICD, Notch target genes as well as bymutation status of Notch receptor and other components of the Notchpathway.

The present invention also provides a method for treating and/orpreventing cancers, said method comprising administering the6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), its derivatives, or thepharmaceutical composition of the invention to a subject in needthereof.

In another embodiment, the present invention provides a method oftreatment of a disease associated with an up-regulated Notch signallingpathway activity, said method comprising administrating the6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), a derivative thereof, orthe pharmaceutical composition of the invention to a subject in needthereof.

The daily dose of compounds of the present invention will necessarily bevaried depending upon the host treated, the particular route ofadministration, and the severity and kind of the illness being treated.Accordingly the optimum dosage may be determined by the practitioner whois treating any particular patient. Further, it is noted that theclinician or treating physician will know how and when to start,interrupt, adjust, or terminate therapy in conjunction with individualpatient response.

For any compound used in the method of the present invention, atherapeutically effective dose can be estimated initially from cellculture assays, animal models, or microdosing of human subjects.

“Treatment” as used herein, refers to both therapeutic treatment andprophylactic or preventative measures. Subjects in need of treatmentinclude those already with the disorder, such as cancer, as well asthose in which the disorder, such as cancer, is to be prevented. Hence,the mammal, preferably human, to be treated herein may have beendiagnosed as having the disorder, such as cancer, or may be predisposedor susceptible to the disorder, such as cancer.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of tumor or cancer cells, reduce the tumor size; inhibit(i.e., slow to some extent and preferably stop) cancer cellsinfiltration into peripheral organs; inhibit (i.e., slow to some extentand preferably stop) tumor metastasis; inhibit, to some extent, tumorgrowth; and/or relieve to some extent one or more of the symptomsassociated with the cancer. To the extent the compounds of the presentinvention may prevent growth and/or kill existing cancer cells, it maybe cytostatic and/or cytotoxic. The phrase “therapeutically effectiveamount” is used herein to mean an amount sufficient to prevent, orpreferably reduce by at least about 30 percent, preferably by at least50 percent, preferably by at least 70 percent, preferably by at least 80percent, preferably by at least 90%, a clinically significant change inthe growth or progression or mitotic activity of a target cellular mass,group of cancer cells, or other feature of pathology.

Optionally the compounds of the present invention may be used againstcell proliferate diseases in combination (for example either at the sametime, or almost at the same time, or one after the other) withconventional treatments such as standard radiotherapy and/or standardchemotherapy. The standard radiotherapy and chemotherapy can be also theconcomitant chemo-radiotherapy.

Therefore, optionally, the standard radiotherapy and/or chemotherapy canbe performed before, simultaneously or after the administration of atherapeutically effective amount of the compound of the presentinvention, or pharmaceutical compositions containing thereof.

The term “concomitant chemo-radiotherapy” is used when these twotreatments (chemotherapy and radiotherapy) are given either at the sametime, or almost at the same time, for instance one after the other, oron the same day, etc.

The term “standard radiotherapy” refers to the use of ionizing radiationas part of cancer treatment to control malignant cells. Preferably theionizing radiation is γ-irradiation. It is also common to combineradiotherapy with surgery, chemotherapy, hormone therapy, orcombinations thereof. Most common cancer types can be usually treatedwith radiotherapy. The precise treatment intent (curative, adjuvant,neoadjuvant or palliative) will depend on the tumor type, location, andstage, as well as the general health of the subject in need thereof.

The term “standard chemotherapy” generally refers to a treatment of acancer using specific chemotherapeutic/chemical agents. Achemotherapeutic agent refers to a pharmaceutical agent generally usedfor treating cancer. The chemotherapeutic agents for treating cancerinclude, for example, Altretamine, Bleomycin, Busulphan, Capecitabine,Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine,Crisantaspase, Cyclophosphamid, Cytarabine, Dacarbazine, Daunorubicin,Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil,Gemcitabine, Idarubicin, Ifosfamide, Irinotecan, Lomustine, Melphalan,Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin,Pentostatin, Procarbazine, Streptozocin, Taco, Temozolomide,Tioguanine/Thioguanine, Thiotepa, Topotecan, Treosulfan, Vinblastine,Vincristine, Vindesine or Vinorelbine.

When a chemotherapeutic agent is used in combination with a compoundaccording to the present invention, then this may be used in the form ofa medicament containing a combination of these two agents, forsimultaneous administration, or they may be used in the form of separatedosage forms, each containing one of the agents, and in the latter casethe individual dosage forms may be used e.g. sequentially, i.e. onedosage form with the compound of the invention, followed by a dosageform containing the chemotherapeutic agent (or vice versa). Thisembodiment of two separate dosage forms may be conceived and provided inthe form of a kit.

Also optionally the compounds of the present invention may be usedagainst cell proliferate diseases, such as cancers, in combination withconventional removal of a tumor bulk, by for example segmental resection(biopsy or gross resection).

The term “removal of a tumor bulk” refers to any removal, ablation orresection of a tumor bulk from a subject. The removal can be chemical,radiation or surgical. Preferably said removal is surgical, such asablation or resection. Resection can be “segmental resection” (orsegmentectomy), a surgical procedure to remove part of an organ or glandfrom a subject. It may also be used to remove a tumor and normal tissuearound it. Debulking agent may be also used to remove tumor bulk. Theterm “debulking agent” includes any molecule (e.g. chemical, biological)or any external/environmental agent (e.g. γ-irradiation) or traditionalsurgery that would allow killing cancer cells from the tumor bulk (e.g.FL1⁰ and FL1⁻ cells as mentioned above).

Another object of the present invention is a kit comprising one or moredoses of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), or of one of itsderivatives having Notch signalling pathway inhibition properties, orthe pharmaceutical composition of the present invention for use in amethod for treatment and/or prevention of cancers. The kit can furthercomprise one or more doses of a chemotherapeutic agent. Optionally, thekit may also comprise reagents and/or instructions for use.

Generally, the kit comprises a container and a label or package inserton or associated with the container. Suitable containers include, forexample, bottles, vials, syringes, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds the pharmaceutical composition that is effective for treating thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). The label or package insert indicatesthat the composition is used for treating the condition of choice, suchas cancer.

The present invention also relates to the use of the compounds of theinvention for inhibiting in vitro or in vivo the Notch signallingpathway in cells. Usually, said cells are cancer cells.

Also envisioned is a method of treating a subject for Notch dependentcancer, comprising

i) determining in cancer cells obtained from a biological sample of saidsubject whether the cancer is Notch signalling pathway dependent, ii)and treating said subject based upon whether the cancer is Notchdependent cancer by administering a therapeutically effective amount of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I or one of itsderivatives having Notch signalling pathway inhibition properties, or apharmaceutical composition of the invention.

Usually, the Notch signalling pathway dependency in cancer cells isdetermined by any method known in the art. As an example, this methodcan consist in an in vitro γ-secretase complex activity assays asdescribed herein.

This method of treating may further comprise administering at least oneconventional cancer treatment. The conventional cancer treatment isadministered before, simultaneously or after the administration of thetherapeutically effective amount of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I or one of itsderivatives having Notch signalling pathway inhibition properties, orthe pharmaceutical composition of the invention.

Usually, the conventional cancer treatment consists in radiotherapyand/or chemotherapy.

The present invention also relates to the use of the compounds of theinvention in a method for provoking apoptosis in a cell, either in vitroor in vivo, by inducing G0/G1 cell cycle arrest.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications without departing fromthe spirit or essential characteristics thereof. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.The present disclosure is therefore to be considered as in all aspectsillustrated and not restrictive, the scope of the invention beingindicated by the appended Claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each ofwhich is incorporated herein by reference in its entirety.

The foregoing description will be more fully understood with referenceto the following Examples. Such Examples, are, however, exemplary ofmethods of practicing the present invention and are not intended tolimit the scope of the invention.

EXAMPLES Example 1 Constructs and Gene Reporter Assays

Mouse DL4-IRES dsRED cDNA was cloned into a pENTR1 vector (Invitrogen®)and finally shuttled into a destination lentivirus vector using Gatewaycloning strategy (Invitrogen). A Phosphoglycerate kinase (PGK) promoterdrove the expression of DL4 protein. DL4-lentiviral particles wereproduced in 293T cells by cotransfection of DL4-lentivirus vector,Gag/pol expression plasmid and plasmid encoding for viral envelopeproteins. To overexpress Notch1 protein, mouse full length Notch1 cDNAwas a cloned in a pCDNA3.1-IRES-puromycin vector. Notch1 cDNA was clonedupstream of IRES-puromycin between HindIII and XbaI restriction sites. ACMV promoter controlled the expression of Notch1 protein.

To measure the Notch signalling activation, CSL/RBP-jk consensus DNAbinding sequences were cloned in a head to tail conformation in the pGL4luciferase vector (Promega), thus named 12×CSL/RBP-jjk luciferasevector. In order to determine the Notch pathway activation in chemicalcompound screening assay, 12× CSL/RBP-jk consensus DNA binding sequenceswere cloned into pGL4.26.luciferase vector (Promega). As an internalcontrol for transfection efficiency, SV40 Renilla vector was used(Promega).

NICD-GFP overexpression studies were performed using pEGFP-C1-NICDexpression plasmid. The FLAG-CMV2 plasmid expressing MAML1-FLAG was akind gift from Dr. Lizi Wu, Harvard Medical School, Boston.

Generation of DL4 and N1 Stable HeLa Cells:

In order to generate DL4 and N1 stable cell lines, HeLa cell were boughtfrom ATCC (catalog # CCL-2). To generate DL4 stable lines, cells weretransduced with DL4-lentiviral particles. DL4 stable clones wereselected using puromycin. High DL4 expressing clones were sorted usingantibodies against DL4 with Fluorescence activated cell sorting (FACS).To generate N1 stable line, cell were transfected with apCDNA3.1+(Invitrogen) plasmid containing mouse full length Notch1 underthe control of CMV promoter. To select for Notch1 expressing clones, anIRES puromycin cassette was cloned downstream of Notch1 cDNA. HeLa cellsexpressing high levels of Notch1 were enriched by FACS using anti-Notch1antibodies. DL4- and N1-HeLa cells were cultured in DMEM (GIBCO,Invitrogen), 10% FCS and 10 ug/ml of puromycin (Sigma).

High-Throughput Screening and Data Analyses:

For chemical library screen, the coculture assay was performed asfollows. N1-HeLa cells were cotransfected in 10 cm tissue culture disheswith 16 μg of pGL4.26.12×CSL.luciferase vector/plate, 4 μg of Notch1expression plasmid/plate, and 200 ng of SV40 Renilla vector/plate. DL4-and N1-HeLa cells were detached from the plate by using 0.5 mM EDTA(1×PBS). The both cell populations were counted and mixed in 1:1 ratio(5,000:5,000 cells/well in a 384 well plate) and dispensed into 384 wellplates (white, clear bottom, Corning) using multidrop Combi platedispenser. The assay plates were pre-dispensed (using automated Biomek3000 liquid handler) with chemical compound libraries (MicrosourceNIMDS, Maybridge Hitfinder and Prestwick) to give a final concentrationof 10 μM. The final assay volume was 22 μls. Twenty-four hours later,growth media was aspirated and cells were lysed with 1× Passive lysisbuffer for 10 minutes at room temperature. Luciferase activity wasmeasured using Luciferase Assay Reagent II and Renilla values weredetermined by using Stop and Glow reagent (Dual luciferase assay system,Cat # E1980, Promega). Luciferase and renilla readouts were taken usingTecan® F500 (Tecan) multiplate reader. All the liquid handling steps(aspiration of the medium, dispensing of Passive lysis buffer,Luciferase Assay Reagent II and Stop and Glow reagents) were performedusing ELF406 liquid handler.

Data analyses were performed using in-house built analyses software atBiomolecular Screening Facility (BSF) at Ecole Polytechnique Federale deLausanne (EPFL).

RNA Extraction

Total RNA was extracted from cells using TRIzol® extraction kit(Invitrogen). Briefly, 1×10⁶ cells were washed with ice-cold 1×PBS andlysed in 1 ml of TRIzol® solution for 5 minutes at room temperature todissociate nucleoprotein complexes. Lysed cells were then treated with200 μl of chloroform and shaked vigorously for 15-30 seconds andincubated at room temperature for 2-3 minutes. The samples werecentrifuged at 14000 rpm using Eppendorf table top centrifuge for 10minutes at 4° C. Following centrifugation, upper aqueous phase wastransferred to new eppendorf tubes. To precipitate total RNA 500 μl ofisomyl alcohol was added to the separated aqueous phase and incubated atroom temperature for 10 minutes. A RNA pellet was obtained bycentrifuging the samples at 4° C. for 10 minutes. RNA pellet obtainedwas washed with 1 ml ice cold 75% ethanol and spun down at 14000 rpm at4° C. RNA pellet was dried off of excess of ethanol and resuspended in40 μl DPEC water.

cDNA Synthesis:

Total RNA extracted from the cell was used to synthesize cDNA by reversetranscription reaction. Reverse transcription was performed usingSuperScript™ RT (Invitrogen). RNA concentration was measured usingNanoDrop®ND-1000 spectrophotometer (Witec AG) and 500 ng of total RNAwas mixed with a 10 mM mix of dNTPs and 100 ng of random primers. Thereaction mix was incubated at 65° C. for 5 minutes and quickly incubatedon ice for 1 minute. Following incubation on ice, 5× first strand bufferand 0.1M DTT were added and mix was incubated for 2 minutes at 25° C. Tostart the reverse transcription reaction, 200U of SuperScrip™ II RT wasadded to the reaction mix and incubated at 42° C. for 50 minutes. Thereaction was stopped by incubating the reaction mix at 75° C. for 15minutes.

Western Blot Analyses:

Cells were lysed in RIPA buffer (50 mM Tris.Cl, pH 7.5, 150 mM NaCl, 1%nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS) for 30 minutes at4° C. Lysed cells were centrifuged to remove the debris at 14000 rpm at4° C. Supernatant was transferred to a new eppendorf tube. The proteinconcentration was determined by Bradford assay using spectrophotometer(Ultrospec 3000 pro). 40 μg of protein were denatured in 1×SDS gelloading buffer (100 mM Tris.Cl, pH 6.8, 200 mM DTT, 4% SDS, 0.2%bromophenol, 20% glycerol) by heating at 99° C. for 5 minutes. Denaturedprotein samples were stored on ice until loading on to the acrylamidegel. The samples were run on 8% or 10% acrylamide gel in Tris-glycineelectrophoresis buffer (25 mM Tris, 250 mM glycine, 0.1% SDS). Followingseparation on the acrylamide gel, protein samples were transferred on toPVDF membrane (PEQ lab, catalog number 39-3010) using transfer buffer(39 mM glycine, 48 mM Tris base, 0.037% SDS and 20% methanol).

For immunoblotting, membranes were blocked with 5% milk and incubatedovernight with primary antibodies at 4° C. Membrane were washed with1×TBST (1×TBS+0.5% tween 20) for 5 minutes (3 times) and incubated withHRP-conjugated secondary antibodies for one hour at room temperature.Signal was detected with Super Signal West chemiluminescent substrate(Thermo Scientific, catalog number 34077).

Immunofluorescence Staining:

To perform immunofluorescence staining, HeLa cells or C2C12 cells weregrown on cover slips. Cells were washed with 1× ice-cold PBS, fixed with4% PFA for 5 minutes at room temperature and permeabilized using 0.3%Triton X-100. Subsequently permeabilized cells were blocked for 20minutes with 1% BSA for 20 minutes at room temperature. Cells wereincubated with appropriate primary antibodies for one at roomtemperature. Alexa Fluor-488 conjugated secondary antibodies were usedto detect primary antibodies. Cells were counterstained with DAPI andmounted in fluorescent mounting media. Fluorescent images were viewedand captured using Zeiss Axioplan microscope at Bioimaging and opticscore facility at EPFL.

TABLE 1 List of the antibodies and working dilutions. AntibodiesApplication Dilution Source Anti-Val 1744 NICD WB and ChIP 1:1000 CellSignal, 2421S Anti-Notch1 (C-20) WB 1:1000 Santa Cruz, sc-6014Anti-RBP-jk IF 1:500 Santa Cruz, sc-28713 Anti-FLAG-M2 IF 1:500 Sigma,F1804 Anti-Hes1 (H-140) WB 1:500 Santa Cruz, sc-25392 Anti-cMyc (9E10)WB 1:500 Abcam, ab11917 Anti-Delta like 4 Flow cytometry 1:100 Producedin-house Anti-Notch1 Flow cytometry 1:50 Produced in-house Anti-TubulinWB 1:3000 Sigma, Anti-Myosin heavy chain IF 1:200 Sigma, MY-32HRP-conjugated anti-goat IgG WB 1:3000 Invitrogen, 611620 HRP-conjugatedanti-mouse IgG WB 1:3000 GEhealthcare, NA931V HRP-conjugated anti-rabbitIgG WB 1:3000 GEhealthcare, NA934V Alexa Fluor-488 secondary Ab IF1:1000 Invitrogen Anti-B220-Pacific blue Flow cytometry 1:400 Producedin-house Anti-CD21-FITC Flow cytometry 1:200 eBioscience Anti-CD23-PEFlow cytometry 1:400 BD Pharmingen Anti-TCR β-APC eF780 Flow cytometry1:400 eBioscience Anti-CD4-FITC Flow cytometry 1:800 Produced in-houseAnti-CD8-Alexa 648 Flow cytometry 1:600 Produced in-house Anti-CD71-PEFlow cytometry 1:800 eBioscience Anti-Ter119-APC eF780 Flow cytometry1:200 eBioscience Anti-AnnexinV-Cy5 Flow cytometry 1:50 BD Pharmingen

C2C12 Myoblast Differentiation Assay:

C2C12 cells were grown on collagen-coated cover slip in the presence ofgrowth media (10% serum). To induce myoblast differentiation, cells weregrown to 100% confluency for 3 days in the presence of differentiationmedia (2% horse serum) or in the presence of growth media+Notchinhibitors. After 3 days, cells were washed with 1× ice-cold PBS andfixed with 4% PFA. Immunofluorescence staining was performed usinganti-MHC antibody as explained in the section 2.2.6 (Immunofluorescencestaining)

Flow Cytometry Analyses:

Fluorescence activated cell sorting (FACS) analyses were performed onCyAn™ ADP instrument platform for flow cytometry at Flow cytometry corefacility, EPFL. DL4 and Notch1 expression in DL4- and N1-HeLa cells wasdetermined using anti-DL4 and anti-N1 antibodies respectively. T celldevelopment in the thymus was investigated using antibodies against CD4,CD8 and TCRβ. MZB cell development was monitored using antibodiesagainst B220, CD21 and CD23. In brief, a single cell suspension wasprepared from thymus and spleen. 1×10⁶ cells suspended in 50 μl ofstaining media (HBSS supplemented with 2% NCS and 25 mM HEPES) andstained with appropriate antibody combinations by incubating on ice for30 minutes.

To quantitate the percentage of apoptotic cells, AnnexinV and 7AADstaining was performed. Thymic cells were suspended in 300 μl of 1×AnnexinV binding buffer (BD Biosciences, San Diego, USA) and incubatedwith 10 μl of AnnexinV-Cy5 antibody and 10 μl of 7AAD (BD Biosciences,San Diego, USA). Samples were incubated for 15 minutes at roomtemperature. FACS was performed with in one hour of antibody staining.

Flow cytometry analyses were done on live cells by gating on forwardscatter (FSC) and side scatter (SSC). Data were analyzed by FlowJosoftware (Tree Star, Ashland, Oreg.).

Alamar Blue Proliferation Assay:

Alamarblue® proliferation assays were performed to determine the growthkinetics of Notch inhibitor treated cells. Alamar Blue® consists of acell permeable substrate resazurin. In metabolically active andproliferating cells, resazurin is converted to resorufin due to anintrinsic reducing power of live cells and produces a red fluorescence.Therefore production of resorufin serves as an indicator of theviability of the cell population.

Proliferation assays were performed by seeding 5000 cells/well in a 96well plate. Cells were treated with DMSO or Notch inhibitors fordifferent time intervals. Each treatment for every time interval wascarried out in 8 replicates. To determine the growth kinetics, 10 μl ofAlamar Blue® (Invitrogen) was added to each well and incubated for 4hours. Alamarblue readout was taken using Tecan F500 (Tecan) multiplatereader.

Haematoxylin & Eosin Staining:

Organs were harvested, fixed in 4% paraformaldehyde (PFA) overnight at4° C. and embedded in paraffin. Tissue sections were dewaxed andhydrated using decreasing concentration of ethanol (100%-70%) andfinally in distilled water. Sections were stained with Hemotoxylin for 5minutes, rinsed in acid alcohol for about 20 seconds and then rinsed inrunning water for 10 minutes. Sections were then stained with Eosin for5 minutes, washed in water and dehydrated using increasing concentrationof ethanol (70%-100%) and cleared in xylene solution. Sections were themounted using mounting solution. Haematoxylin and Eosin stained sectionswere viewed and images were captured using Leica DMI4000 microscope.

Alcian Blue Staining:

Intestinal tissue was flushed with ice-cold 1×PBS, fixed in 4% PFA.Tissues were embedded in paraffin and sectioned to a thickness of 4microns. Intestinal sections were deparaffinized at 60° C. and hydratedwith decreasing concentration of alcohol (100%-70%) and finally washedin distilled water. Alcian blue staining was performed for 30 minutes atroom temperature washed in running water and finally counterstained innuclear fast red solution for 5 minutes. Tissue sections were thenwashed in running water dehydrated in 100% alcohol and cleared in xylenesolution. Mounted sections were then viewed and images were capturedusing Leica DMI4000 microscope.

Experimental Mice:

Mice were kept and bred at Animal facility, EPFL, Lausanne. C57B16 micewere used to assess the intestinal toxicity of the chemical compounds.MMTV-ErbB2/Neu-IRES Cre (FVB background) mice were obtained from Dr.William J Muller, McGill University, Montreal and genotyped usingMMTV-ErbB2/Neu specific primers (Ursini-Siegel et al., 2008).NOD/SCIDγc^(−/−) mice were bought from The Jackson Laboratory (USA) andwere kept and bred at Animal facility, EPFL, Lausanne.

Intestinal Toxicity and Effect on Marginal Zone B Cell Development:

C57B16 mice were intra peritoneal (i.p) injected with oil or 25 mg/kg ofI3 or 10 mg/kg of CPA, once a day for 5-7 days. Mice were weighed usinga weighing scale on day 0, day 3 and day 5. On day 8, intestinal tissue,spleen and thymus were harvested for analyses.

Tumor Transplantation Assay:

The human leukemic cell lines RPMI 8402 and HPB ALL were transduced witha lentivirus containing luciferase gene constitutively expresseddownstream of a CMV promoter. The human leukemic cell lines RPMI 8204(0.5-1×10⁶ cells) and HPB ALL (1×10⁶) were suspended in 100 μl ofice-cold 1×PBS and kept on ice until the transplant. NOD/SCIDγc^(−/−)mice were transplanted with the human leukemic cell lines by intravenous(i.v) injection. Mice were monitored for tumor development using CaliperIVIS (Xenogen) live imaging system. Briefly, the luciferase substrateluciferin (Biosynth, L-8820) was dissolved in 1×PBS and was injected(intra peritoneal) into the mice at a concentration of 150 mg/kg of bodyweight. Mice were imaged 5 minutes after the luciferin injection usingCaliper IVIS live imaging system.

At day 13-15, mice were treated with oil or 25 mg/kg of I3 on a dailybasis. Images were captured at the end of the experiments.

Primary MMTV-ErbB2/Neu mammary tumors were harvested from the mice and asingle cell suspension was prepared. 1×10⁶ primary tumor cells weresuspended in 50 μl of 1×PBS and kept on ice. Three weeks old recipientFVB mice were cleared of their endogenous epithelium and tumor cellswere injected into the empty fat pad. Tumor development in the recipientmice was monitored and tumor volumes were measured using digitalcaliper. Tumor volumes were calculated using following formula:2×length×(width). Once the tumor reached a volume of about 100 mm³,recipient mice were treated with oil or 25 mg/kg of I3 on alternatedays.

Assay Development

In order to identify novel modulators of the Notch pathway, Applicantshave established a coculture assay in which DL4 ligand expressing HeLacells were cultured with N1 HeLa cells, thereby activating the Notchpathway. The use of a DL4 and N1 HeLa cell coculture system mimicsphysiological conditions of cell-cell communication between ligand andreceptor expressing cells. The in vitro generation of a controlledreceptor-ligand assay system allowed Applicants to modify and monitorthe Notch signal intensity by γ-secretase inhibitors.

DL4: N1 HeLa Cell Coculture Activates Notch Signalling

To set up a coculture assay, Applicants have established DL4 and N1expressing stable HeLa cell lines. In brief, HeLa cells were transducedwith a lentivirus containing DL4 cDNA downstream of the PGK promoter.The D4 expressing cell population was enriched by fluorescence activatedcell sorting. Similarly, the N1 stable HeLa cell line was establishedusing a plasmid containing the mouse N1 cDNA followed by an IRESPuromycin selection cassette. This system allowed Applicants to selectfor only Notch1 expressing clones when selected using puromycin. Theexpression levels of DL4 and N1 proteins in the respective cell lineswere detected using anti-DL4 and anti-N1 antibodies. Quantification ofthe protein levels by flow cytometry showed high level expression of DL4and N1 compared to parental HeLa cells (data not shown).

To assess the Notch pathway activation potential of the stable celllines, DL4 and N1 stable HeLa cells (DL4-HeLa and N1-HeLa, respectively)were cocultured in 1:1 ratio in a 6-well plate and grown to confluency.The cocultured cells were treated with DMSO or DAPT (10 μM) for 24hours. For comparison, parental HeLa cells were also cocultured withDL4-HeLa cells and were grown in the presence or absence of DAPT for 24hours. Western blot analyses for the active form of Notch1 (NICD) usingVAL1744 antibodies was performed and revealed only modest levels of NICDwhen parental HeLa cells were cocultured with DL4 HeLa cells (data notshown), accounting for a low level of endogenous Notch1 in HeLa cells.On the other hand, in the absence of ligand (DL4-HeLa cells) or in thepresence of GSI (DAPT) NICD levels were not detected, indicating a lossof Notch signalling (data not shown). However, cocultures of DL4- andN1-HeLa cells revealed significantly higher levels of NICD, which can beblocked by DAPT treatment (data not shown). Transient introduction offull length Notch1 cDNA into N1-HeLa cells further enhanced therobustness of the coculture assay as indicated by the increased levelsof NICD protein (data not shown). Inhibiting the N1 cleavage with DAPTcan abrogate the increase in Notch signalling activity (data not shown).These results confirmed that high levels of the Notch pathway activationcould be achieved in the DL4:N1 coculture assay that responds to GSIinhibition.

The establishment of DL4:N1 coculture assay in a 6 well plate formatallowed Applicants to assess receptor-ligand interaction mediated Notchsignalling activation. GSI (DAPT) treatment of the coculture system canblock receptor-ligand interaction driven Notch signalling.

Establishment of High-Through Put Screening (HTS) Compatible Assay

Initially, DL4:N1 coculture assay was established in a 6-well plate. Inorder to set up a high-through put screen (HTS), the assay system wasfurther optimized to robustly work in a 384 well plate format.

The assay was scaled-down to a 384 well plate format for screening ofchemical compound libraries. In order to accomplish this, N1-HeLa cellswere transfected with a reporter plasmids and N1 expression vector.Twelve hours later, chemical compounds were dispensed into a 384 wellplate along with DMSO and DAPT as negative and positive controls. DL4-and N1-HeLa cells were mixed in a 1:1 ratio (5000:5000 cells/well) andadded to 384 well plates using multidropCombi plate dispenser.Luciferase readout was measured using dual luciferase assay system. Tooptimize and determine the reproducibility of the assay, half of theplate was treated with DMSO (192 wells) and the second half was treatedwith 10 μM DAPT (192 wells). DAPT treatment led to a 10-folddownregulation of Notch signalling activation. The Z′ value for thisassay was higher than 0.5. A Z′ value of >0.5 confirms the reliabilityand reproducibility of the assays for a HTS campaign.

Example 2 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) as a novel Notchsignalling inhibitor I3 Inhibits NICD Mediated Activation of NotchSignalling:

To validate the Notch inhibitory activity and determine the IC50 valueof the I3 compound, the DL4:N1 coculture assay system was used. Thecells in the coculture assay were treated for 24 hours with anincreasing concentration of I3 (2-10 μM). The activation of the Notchpathway was measured using a Notch driven luciferase reporter assay. Asshown in FIG. 1A, I3 blocks Notch signalling in a concentrationdependent manner with an IC50 value in the lower μM range.

To determine whether over expression of NICD can rescue I3 mediatedinhibition of Notch signalling, HeLa cells were co-transfected with aNICD expression plasmid and 12×CSL luciferase construct. The transfectedcells were treated with an increasing concentration of I3 and DAPT.Surprisingly, treatment of NICD expressing cells with I3 could blockpathway activation in a dose-dependent manner, while DAPT had no effecton the signalling activation (FIG. 1B). This data suggests that I3mediated inhibition of the Notch pathway is due to its activitydownstream of the S3 cleavage event.

Next Applicants investigated whether I3 could block pathway activationvia other Notch receptors or is it specific to Notch1 signalling. Toaddress this, a coculture assay was used where Notch signalling wasactivated via DL4:N1 or DL4:N2 ligand receptor pairs. The treatment ofcells in these two coculture assays with I3 caused an inhibition ofNotch signalling via both DL4:N1 and DL4:N2 ligand-receptor pairs (FIG.1C). Similarly, I3 could also block pathway activation byNotch1-intracellular domain (NICD) and Notch2-intracellular domain(N2-ICD), suggesting that I3 is not specific for NICD mediatedactivation (FIG. 1D).

6-(4-Tert-Butylphenoxy)Pyridin-3-Amine does not Block NuclearLocalization of NICD:

In vitro data from NICD transfected HeLa cells suggested that6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) blocks Notch signalling byacting downstream of S3 cleavage event. This raises severalpossibilities about the mechanism of action of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3). For example,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment could impairnuclear localization of the NICD. A second possible mechanism ofinhibition could be targeting of one or more individual components ofthe transcriptional activation complex in the nucleus. To test whether6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) has an impact on nucleartransport of NICD, HeLa cell were transfected with a NICD-GFP fusionconstruct and treated with DMSO and6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3). This allowed Applicants tofollow transport of fusion protein within the cell. In parallel, the6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) mediated pathway inhibitionwas determined by Notch driven luciferase measurement (data not shown).Microscopic studies showed that in DMSO treated cells NICD-GFP fusionprotein translocate to the nucleus, which was not perturbed upon6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment (data not shown).This data rule out nuclear exclusion of NICD as a mechanism of action of1644-Tert-Butylphenoxy)Pyridin-3-Amine (I3).

Over Expression of MAML1 Above a Certain Threshold can Rescue6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) Induced Notch SignallingInhibition:

As 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) mediated blockage ofNotch signalling does not involve nuclear exclusion of NICD, Applicantsaddressed whether 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) blocksinteraction and thereby sub-nuclear localization of NICD, MAML1 andCSL-RBP-jk (all parts of the core transcriptional activation complex).To resolve this, HeLa cells were co-transfected with 800 ng of NICD-GFPplasmid, 1 μg of MAML1-FLAG expression vector and grown on cover slips.The transfected HeLa cells were treated with DMSO or6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) (10 μM) for 24 hours. Theability of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) to block Notchactivation at this concentration of NICD and MAML1 was verified by Notchdriven luciferase measurement (data not shown). Following treatment, thecells were fixed with 4% PFA, blocked with 1% BSA and stained withantibodies against FLAG tagged MAML1 and CSL-RBP-jk. NICD-GFP fusionprotein was visualized by tracing the GFP protein. When expressed alone,NICD-GFP protein was localized in the nucleus in a diffused manner and6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment did not alter itsnuclear localization. However overexpression of NICD-GFP and MAML1 ledto co-localization of both proteins into sub-nuclear compartments(possibly nuclear bodies). The 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) treatment of the cells did not perturb the translocation andco-localization of NICD-GFP and MAML1 into sub-nuclear compartment (datanot shown).

Similarly, the location of CSL/RBP-jk was investigated using antibodiesspecific against this protein. As shown in FIG. 22C, MAML1-FLAG andCSL/RBP-jk co-localized in sub-nuclear compartments and6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment did not perturbtheir distribution in the nucleus (data not shown). This data suggestthat 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment does notperturb co-localization of components of the Notch transcriptionalactivation complex in the nucleus. However, whether it blocksinteraction between various components of the complex still need to beinvestigated.

To further investigate the mechanism of action of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), Applicants speculated that6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) might be targeting one ofthe components of the transcriptional activation complex. The overexpression of this target protein may titrate out6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) compound and thus rescue6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) induced pathway inhibition.To address this question, HeLa cells were co-transfected with 800 ng ofNICD-GFP plasmid and with an increasing amount (0, 1 and 3 μg) ofMAML1-FLAG expression vector. The Notch pathway activation was measuredby introducing 12×CSL luciferase plasmid. As shown in FIG. 2, in HeLacells transfected with NICD alone or NICD+1 μg of MAML1,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment can block Notchsignalling in a concentration dependent manner. However, when the amountof MAML1 plasmid was increased to 3 μg,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment was no longer ableto inhibit the activation of Notch signalling (FIG. 2). Therefore, overexpression of MAML1 above a certain threshold could rescue6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) mediated inhibition of theNotch pathway. This data suggest that MAML1 itself might be the targetof 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) compound. An increase inthe concentration of MAML1 may be able to titrate out the inhibitor andthus rendering it incapable of blocking the signalling cascade.

6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) Treatment Decreases NotchSignalling in Human Cancer Cell Lines:

Aberrant activation of Notch signalling plays an important role in tumorinitiation and/or maintenance of human cancers. To determine whether6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment can block Notchsignalling in human cancer cells, various cancer cell lines (T-ALL celllines RPMI 8402, HPBALL, KOPTK1 and pancreatic cancer cell line PANC1)were treated with 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) for 24hours. The effect on Notch signalling was determined by measuring theexpression levels of Notch target genes.6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment of human cancercell lines (RPMI 8402, HPBALL, KOPTK1 and PANC1) for 24 hours andsubsequent analyses of Notch target genes by qRT-PCR or Western blotanalyses showed that 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) induceda statistically significant downregulation of Notch target genes such asHes1, cMyc and Dtx1 at the mRNA as well as at the protein levels (FIG.3). The downregulation of Notch target genes correlates with reducedlevels of NICD (FIGS. 3B, C and D).

As treatment of the human T-ALL cell lines and PANC1 pancreatic cancercell line with 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) induced adownregulation of Notch signalling, Applicants questioned whether thisinhibition of the pathway translates into growth arrest in cancer cells.To this end, RPMI 8402, KOPTK1, PANC1 and nRas driven melanoma celllines were grown in the presence or absence of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) for several days and theirproliferative index was measured using the Alamar blue assay. Inaddition, B-lymphocyte RAJI cell lines with no known Notch mutationswere used as a control (data not shown). As shown in FIG. 4,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) and DAPT treatment induced asignificant proliferation block in T-ALL cell lines RPMI 8402, KOPTK1and pancreatic cancer line PANC1. Similarly,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) significantly inhibited thegrowth of nRas driven melanoma cell lines (FIG. 4). However neither DAPTnor 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) had any effect on theproliferation of Notch-independent RAJI cells (data not shown).

6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) but not DAPT Blocks NotchSignalling in NICD Overexpressing Human T-ALL and Breast Cancer CellLine.

6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) can block NICD mediatedactivation of the Notch pathway (FIG. 1). To determine whether6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) can also induce aproliferation block in NICD overexpressing cells, the human T-ALL cellline DND41 (DND41-Parental) was transduced with a NICD expressinglentivirus to generate DND41-NICD cell line. These two cell lines weretreated with DMSO, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) and DAPT.The treatment of DND41-parental cell line with DAPT and6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) led to a downregulation ofHes1 when compared to DMSO treated cells. However, when DND41-NICD cellswere treated with DMSO, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) andDAPT, only 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), but not DAPTtreatment caused a downregulation of Hes1 (FIG. 5A). In addition, thesetwo cell lines were also monitored over several days foranti-proliferative effects of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) and DAPT. It was observed that while both6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) and DAPT treatment caused asignificant proliferative block in the DND41-Parental cell line (FIG.5B), only 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) was able to inducea growth arrest in DND41-NICD cells (FIG. 5C). This data furtherstrengthen the notion that the 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) compound can block NICD mediated pathway activation andproliferation in human cancer cells.

To further strengthen the notion that6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) can block NICD mediatedpathway activation and proliferation of human cancer cells, HCC1187human breast cancer cell lines was treated with this compound. HCC1187cell line harbors a SECC22B-Notch2 chromosomal translocation, thusgenerating constitutively active form of N2-ICD (FIG. 5D). Due to thismutation, HCC1187 cell lines do not respond to γ-secretase inhibitorssuch as DAPT. As shown is FIG. 5E, while DAPT treatment did not inhibitproliferation of HCC1187 cell lines,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment significantlyinduced a proliferation block in these cell line.

6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) Induces G0/G1 Cell CycleArrest and Apoptosis in Human T Cell Acute Lymphoblastic Leukemia CellLines:

As shown in FIGS. 4 and 5, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3)treatment of human leukemic cell lines and human breast cancer celllines negatively regulate proliferation. This6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) mediated proliferativearrest could be due to induction of apoptosis or cell cycle arrestduring different phases of cell cycle. In addition, inhibition of Notchsignalling using γ-secretase inhibitors has been shown to induce G0/G1cell cycle arrest in human TALL cell lines. Therefore to furtherelucidate the mechanisms responsible for6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) mediated proliferativearrest cell cycle and apoptosis analyses were carried out. Human TALLcell lines (RPMI8402, KOPTK1, TALL1, CUTL1 and HPB ALL) and human breastcancer cell line HCC1187 were treated with6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) or DMSO for 2 days or 7days. To investigate cell death, Annexin V staining was performed andproportion of apoptotic (AnnexinV positive) cell population wasdetermined by flow cytometry analyses after 7 days of treatment. Asshown in FIG. 6A, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatmentinduces significant apoptosis in RPMI8402, CUTL1, KOPTK1, TALL1 and HPBALL. Similarly, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine induces apoptosisin human breast cancer cell line HCC1187 (FIG. 6C).

In addition to induction of apoptosis, the proliferative arrest observedin 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treated human leukemiccell lines and breast cancer cell line also appears to be due to cellcycle arrest in G0/G1 phase of the cell cycle. Leukemic cell lines(RPMI8402, KOPTK1 and TALL1) and breast cancer cell lines were treatedwith 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) for 48 hours and cellcycle status was determined using Ki67 and Hoechst stain. As shown inFIGS. 6B and 6D, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) induces anarrest in the G0/G1 phase of the cell cycle, a phenotype normallyobserved due to inhibition of Notch signalling.

6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) Mediated Notch SignallingInhibition Induces C2C12 Myoblast Differentiation:

To further confirm the Notch inhibitory potential of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) in different systems, C2C12myoblast differentiation was used as a functional assay. The Notchpathway activation in C2C12 myoblasts retains them in anundifferentiated state, while abrogation of Notch signalling inducestheir differentiation. C2C12 myoblasts were treated with DMSO,6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) and DAPT and grown to 100%confluency for 3 days. After three days, cells were fixed and stainedwith antibodies against Myosin Heavy Chain (MHC) protein. Cell nucleiwere counterstained with DAPI. C2C12 myoblasts grown in the presence of10% serum (growth medium) maintain their undifferentiated state, whilethe cells treated with DAPT and 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) started to differentiate into multinucleated MHC positive myotubes(data not shown).

6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) does not Impede Upon Wnt andHedgehog Signalling Cascades

One of the concerns about the activity of the chemical compound6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) is its specificity towardsthe Notch signalling pathway. In order to test whether6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) could also block otherdevelopmental pathways, Applicants have tested its ability to block theWnt and Hedgehog signalling pathways. In summary, to measure Wntsignalling, HeLa cells were transfected with a plasmid containing apromoter consisting of TCF/LEF binding sites and thereby driving theexpression of a luciferase gene (TOP-luciferase). To activate the Wntpathway, a plasmid encoding for β-catenin was co-transfected into HeLacells. The co-transfected cells were incubated in the presence orabsence of the 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) chemicalcompound. Transient introduction of β-catenin leads to an upregulationof Wnt signalling as measured by β-catenin-TCF/LEF driven luciferaseactivity. Importantly, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3)treatment of cells with activated Wnt signalling does not block the Wntpathway activation (data not shown).

Using a similar strategy, Hedgehog signalling was activated in HeLacells by introducing the Gli1 transcription factor and pathwayactivation was monitored using a promoter sequence containing Gli1binding sites driving the luciferase expression. The treatment of thesecells with 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) did not inhibitthe Hedgehog signalling cascade (data not shown). Taken together thesedata suggest that the 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3)chemical compound may not impair other developmental pathways and mightbe specific for Notch signalling inhibition. However it still needs tobe determined whether the resistance of Wnt and Hedgehog signallingtowards 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) is cell typespecific or whether it is a general phenomenon.

In Vivo Effects of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) in C57BL6Mice:

Notch signalling regulates homeostasis of several organs duringdevelopment. For example, Notch1 mediated pathway activation isessential for T cell development in the thymus (Radtke et al., 1999).However, the Notch1 driven T cell development does not appear to bedependent on MAML1, as the loss of MAML1 did not perturb T celldevelopment in the mice. This could be due to a compensatory mechanismby MAML2 and MAML3 family members for the loss of MAML1. In the spleen,Notch2 driven signalling exclusively via MAML1 is required for MZB celldevelopment. Genetic ablation loss of Notch2 and MAML1 cause a block inthe development of MZB cells (Wu et al., 2007, Saito et al., 2003). Inaddition, Notch signalling via both Notch1 and Notch2 is essential forthe maintenance of the crypt compartment. A compound genetic ablation ofNotch1 and Notch2 in the intestine leads to goblet cell metaplasia.Applicants therefore, investigated whether6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) could impair above-mentionedNotch-dependent developmental processes.

In in vitro culture assays, chemical compound6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) was able to block Notch1 andNotch2 mediated pathway activation. Therefore, Applicants hypothesizedthat treatment of mice with 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3)may lead to a goblet cell metaplasia of the intestine. To test thishypothesis, mice were intra peritoneally (i.p) injected with 25 mg/kg of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) for 7 consecutive days. Onday 8, animals were sacrificed and intestinal tissues were fixed andembedded in paraffin. Histological analyses were carried out usingAlcian blue to stain for goblet cells. Surprisingly, despite its abilityto block both Notch1 and Notch2 mediated pathway activation in in vitrocultures, the intestinal tissue of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treated mice was completelynormal with intact architecture and no indication of goblet cellmetaplasia (data not shown). Similarly, the effect of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) on body weight changes wasalso monitored. Mice were injected for 5 consecutive days with 25 mg/kgof 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) and the changes in bodymass were recorded. The treatment of mice with6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) did not cause a loss in thebody weight.

6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) Treatment Induces a Block inMZB Cell Development:

Applicants hypothesized that 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3)mediated inhibition of Notch2 signalling should lead to a block in MZBcell development in the spleen. MZB cell development was assessed byflow cytometry staining of splenocytes with antibodies directed againstB220, CD21 and CD23. As shown in FIG. 7B treatment of mice with6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) leads to a reduction in thepercentage of MZB cell population in the spleen. In addition, the lossof MZB cell population in the spleen also reflects in the absolutenumber of MZB cells (FIG. 7C).

Therefore, 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) mediated block inMZB cell development mimics loss of Notch2 and MAML1 phenotype. However,it is still need to be seen, whether6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) exert its Notch inhibitoryeffect only via MAML1 or it could block Notch signalling via other MAMLfamily members as well.

6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) Treatment Slows Tumor Growthof Human T Cell Leukemia in a Xenotransplantation Model:

Activation of Notch signalling due to activating mutations in differentcomponents of the pathway are known to cause more than 50% of the humanT cell acute lymphoblastic leukemias. Therefore, Applicants decided toinvestigate the anti-cancer activity of the chemical compound6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) in Notch driven human T cellleukemia in vivo. To achieve this goal, xenotransplant models of humanleukemia were established using NOD/SCID γc^(−/−) mice. Human T-ALL celllines HPB ALL and RPMI 8402 were used for this purpose. The HPB ALL cellline harbours a L1575P mutation in the heterodimerization domain and aninsertion in the PEST domain of the Notch1 receptor, therebyconstitutively activating the Notch1 signalling. Similarly, RPMI 8402cells exhibit ligand independent Notch signalling activation due to aninsertion at 1584 a.a residue in the heterodimerization domain and alsoan inactivating mutation (R465H) in the E3 ligase FBW7. Both these cellsline were found to respond to 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) treatment in in vitro culture assays in terms of proliferationand/or downregulation of the Notch target genes. To determine whetherthese cell lines establish leukemia in a xenotransplant setting, onemillion cells from each line were intra venously (i.v) injected intoNOD/SCIDγc^(−/−) mice. The animals developed leukemia with 100%penetrance and die within 4 weeks after transplantation.

Once RPMI 8402 and HPB ALL cell lines were shown to develop leukemia ina xenotransplantation assay, they were transduced with a lentivirusconstitutively expressing luciferase gene. This allowed Applicants tovisualize and monitor the leukemia progression in the mice using theCaliper IVIS (Xenogen) live imaging detection system. In order todetermine the anti-cancer efficacy of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) in established tumors, amaintenance experiment was performed. One million HPB ALL cells wereinjected (i.v) into NOD/SCID γc^(−/−) mice. Mice were monitored forleukemia development by detecting luciferase expressing leukemic cells.Once the disease was established around day 15, the mice were split intotwo groups. One group was treated with oil as a control and the secondgroup was treated with 25 mg/kg of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) on a daily basis. As shownin FIG. 8A, the mice treated with oil develop leukemia with 100%penetrance while leukemia in the 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) treated mice did not progress at the same rate as in the oiltreated group (FIG. 8A).

Similarly in a preliminary experiment, NOD/SCID γc^(−/−) mice weretransplanted with 5×10⁵ RPMI 8402 cells and treated with oil or6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) following the establishmentof the disease. As shown in FIG. 8B, animals treated with oil, developedleukemia while 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treated micewere free of the disease. Furthermore, histological analyses revealedthat in oil treated mice, leukemic cells progressed to infiltrate theliver, but 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treated mice didnot develop any metastatic lesions in the liver (data not shown). Sincethese animals were treated with the chemical compound6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) for 27 days, the intestinaltissue was analyzed to detect any toxicity in the gut. Alcian bluestaining of intestinal tissue did not reveal any abnormality in thegoblet cell numbers and intestinal architecture (data not shown).

Taken together Applicants' data from xenotransplantation model for humanleukemia suggest that 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) hasthe ability to slow down disease progression of an already establishedleukemia. Because of its ability to impact tumor progression, I3 may bea suitable candidate for further development as an anti-cancer agent.

MMTV-ErbB2 Mouse Mammary Tumors Exhibit Notch Signalling Activation andEffect of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) on Mammary TumorProgression.

In human breast cancer, high levels of Notch1 and Jagged1 proteinscorrelated with a poor survival of breast cancer patients. In addition,activation of Notch signalling in human breast cancer also facilitatesbone and lung metastasis. Therefore in order to determine anti-cancerpotential of chemical compound 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) in breast cancer, a mouse model of breast cancer was investigated.The mouse mammary tumor virus (MMTV) driven overexpression of ErbB2 isknown to cause mouse mammary tumors. MMTV-ErbB2 transgenic mice developmammary tumors with a latency of about 5-6 months along with thedevelopment of lung metastasis. One of the characteristics of MMTV-ErbB2mouse mammary tumors is the presence of predominantly luminal epithelialcell types. Notch signalling is known to drive luminal celldifferentiation from mouse mammary stem cells. Therefore Applicantshypothesized that the activation of the Notch pathway in MMTV-ErbB2mammary tumors may contribute towards tumorigenesis in part by favouringluminal epithelial cell differentiation. To this end, Applicantsinvestigated the levels of Notch signalling activation inMMTV-ErbB2-IRES-Cre mammary tumors by measuring the levels of Hes1 byWestern blotting. As shown in FIG. 9A, MMTV-ErbB2 driven mammary tumorsexpress very high levels of Hes1 protein compared to age matched normalmammary glands. To investigate the effect of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) on breast cancerdevelopment, MMTV-ErbB2 mammary tumors were harvested from FVB micecarrying the MMTV-ErbB2 transgene. A single cell suspension was preparedand 5×10⁵ tumor cells were injected into an empty fat pad of a recipientFVB mouse. Once palpable tumors had developed, recipient mice weretreated with either oil or 25 mg/kg of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) on alternate days until theend of the experiment. The tumor volume was measured and recorded onregular intervals. Preliminary results showed that the treatment oftumor bearing recipient mice with 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine(I3) caused significant tumor growth retardation when compared to micetreated with oil alone (FIG. 9B). This data showed that6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) has the ability to slow downthe growth of established breast cancer.

6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) Blocks Notch Signalling inPrimary Human T Cell Acute Lymphoblastic Leukemias:

To further investigate the Notch inhibitory effect of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) in a relevant pathologicalcondition, primary human TALL samples were profiled for the activationof Notch signalling. An accumulation of active form of Notch (NICD) wasused as a biomarker for pathway activation. Several primary human TALLexhibited an accumulation of oncogenic NICD and treatment of thesetumors with 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) leads to adownregulation of this protein. Moreover, the downregulation of NICD inthese primary human TALL samples correlates with a proliferative arrest(data not shown). On the contrary, primary human TALL samples that donot show detectable levels of NICD, did not respond to6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) treatment (data not shown).

These data indicates that an accumulation of NICD can be used as abiomarker for Notch pathway activation and predict treatment outcomeusing Notch inhibitor 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3).

Different Derivatives of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3)Exhibit an Ability to Block Notch Signalling Activation in DL4-N1Coculture Assay:

In order to enhance the Notch inhibitory activity as well as efficacy ofparental 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) compound, differentchemical derivatives of I3 were tested in DL4-N1 coculture assay.Screening of more than 40 different chemical derivatives of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) yielded following compoundsfor their ability to block Notch pathway activation in coculture assay(FIG. 10).

-   I3-A). 6-(4-cyclohexylphenoxy)pyridin-3-amine-   I3-B) 6-(4-(tert-Pentyl)phenoxy)pyridin-3-amine (CAS #1036533-91-1)-   I3-C) 4-(4-(tert-butyl)phenoxy)aniline (CAS #56705-89-6)-   I3-D). 6-(4-Butylphenoxy)pyridin-3-amine-   I3-E). 4-(4-(tert-pentyl)phenoxy)aniline (CAS #328032-81-1)-   I3-F). 4-(4-cyclohexylphenoxy)aniline (CAS #70682-64-3)-   I3-G) 6-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)pyridin-3-amine-   I3-H). 6-(3-(tert-butyl)phenoxy)pyridin-3-amine (CAS #1098366-43-8)-   I3-I). 4-(4-(tert-butyl)phenoxy)-3-fluoroaniline (CAS #946785-77-9)-   I3-J). 4-(4-isopropylphenoxy)aniline-   I3-K). 6-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)pyridin-3-amine-   I3-L). 4-(4-cyclohexylphenoxy)-3-fluoroaniline-   I3-M). 3-fluoro-4-(4-(tert-pentyl)phenoxy)aniline-   I3-N). 6-(4-(2-methylpentan-2-yl)phenoxy)pyridin-3-amine-   I3-O). 4-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)aniline-   I3-P). 4-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)-3-fluoroaniline

As shown in FIG. 10, some of these derivatives (I3-A, I3-B, I3-C, I3-E,I3-G, I3-H, I3-M and I3-N) block Notch signalling to comparable levelsto parental compound I3, while derivatives I3-F and I3-I appears to haveenhanced activity

Example 3 Chemical Synthesis of the Derivative and Precusors Thereof4-(2-methylpentan-2-yl)phenol

4-Butyrylphenol (1000 mg, 6.09 mmol, 1.00 eq) was suspended in toluene(25 mL) and DCM (5 mL) and cooled to 0° C. 2M Me₃Al solution in toluene(7 mL, 14.01 mmol, 2.30 eq) was added dropwise whereby the startingmaterial was dissolved. After stirring at room temperature for 15 h, thereaction mixture was again cooled to 0° C. and TMSOSO₂CF₃ (1.1 mL, 6.09mmol, 1.00 eq) was added dropwise. After stirring at room temperaturefor 3 d, the reaction was quenched by pouring the mixture intoice-water. After acidification with 40% H₃PO₄, the product was extractedwith ethyl acetate (3×) and the organic layers were washed withH₃PO₄-acidic sat. aq. NaCl solution. The solvent was removed underreduced pressure at 30° C. The resulting crude product was purified byflash column chromatography (SiO₂; DCM/petrolether 1:1 to 2:1) to givethe title compound as colourless oil (188 mg, with a purity of around90% (by NMR), 0.95 mmol, 15% yield). R_(f)=0.60 (DCM/MeOH 4%). HRMS(ESI) calcd. for C₁₂H₁₇O⁻ [M−H]⁻ 177.1279. found: 177.1284. ¹H NMR (400MHz, CDCl₃) δ 7.24-7.18 (m, 2H, aromatic H), 6.82-6.76 (m, 2H, aromaticH), 5.12 (s, 1H, OH), 1.60-1.52 (m, 2H, C(CH₃)₂CH₂CH₂CH₃), 1.27 (s, 6H,C(CH₃)₂CH₂CH₂CH₃), 1.16-1.01 (m, 2H, C(CH₃)₂CH₂CH₂CH₃), 0.83 (t, J=7.3Hz, 3H, C(CH₃)₂CH₂CH₂CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 153.03, 142.29,127.11, 114.85, 47.35, 37.25, 29.23, 18.09, 14.90.

General Procedure A:

The respective nitropyridines or nitrobenzenes and the particularphenols were dissolved in DMF or DMSO Anhydrous K₂CO₃ was added and thereaction mixture was stirred at room temperature, unless otherwisestated, until complete conversion. The reaction was then quenched by theaddition of H₂O and the product was extracted with EtOAc or Et₂O. Theorganic layers were washed with 1M aq. NaOH solution (1×) and afterwardswith sat. aq. NaCl solution (1×). The solvent was removed to drynessunder reduced pressure at 30° C. The residue was resolved in DCM andfiltered through cotton to remove inorganic salts. The crude product waspurified by flash column chromatography to afford the correspondingtitle compounds (I3-n, I3-nA to I3-nP).

2-(4-(tert-butyl)phenoxy)-5-nitropyridine, I3-n

Following procedure A, 2-chloro-5-nitropyridine (501 mg, 3.16 mmol, 1.00eq) and 4-tert-butylphenol (611 mg, 4.07 mmol, 1.29 eq) were dissolvedin DMF (6.0 mL) Anhydrous K₂CO₃ (654 mg, 4.73 mmol, 1.50 eq) was addedand the reaction mixture was stirred at room temperature for 14 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:100 to 1:50) to afford thetitle compound as colourless solid (820 mg, 3.01 mmol, 95% yield).R_(f)=0.40 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C₁₅H₁₇N₂O₃ ⁺[M+H]⁺273.1234. found: 273.1229. ¹H NMR (400 MHz, CDCl₃) δ 9.06 (d, J=2.8 Hz,1H, aromatic H), 8.46 (dd, J=9.1, 2.8 Hz, 1H, aromatic H), 7.53-7.42 (m,2H, aromatic H), 7.17-7.05 (m, 2H, aromatic H), 7.01 (d, J=9.1 Hz, 1H,aromatic H), 1.35 (s, 9H, C(CH₃)₃). ¹³C NMR (101 MHz, CDCl₃) δ 167.20,150.49, 148.98, 145.26, 140.29, 134.95, 126.99, 120.84, 111.38, 34.72,31.57.

2-(4-cyclohexylphenoxy)-5-nitropyridine, I3-nA

Following procedure A, 2-chloro-5-nitropyridine (300 mg, 1.89 mmol, 1.00eq) and 4-cyclohexylphenol (417 mg, 2.37 mmol, 1.25 eq) were dissolvedin DMSO (6 mL). Anhydrous K₂CO₃ (397 mg, 2.87 mmol, 1.52 eq) was addedand the reaction mixture was stirred at room temperature for 27 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:100 to 1:75) to afford thetitle compound as colourless solid (600 mg, 2.01 mmol, quant. yield).R_(f)=0.35 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C₁₇H₁₉N₂O₃ ⁺ [M+H]⁺299.1390. found: 299.1392. ¹H NMR (400 MHz, CDCl₃) δ 9.06 (d, J=2.9 Hz,1H, aromatic H), 8.46 (dd, J=9.1, 2.9 Hz, 1H, aromatic H), 7.31-7.22 (m,2H, aromatic H), 7.07 (dd, J=8.7, 2.3 Hz, 2H, aromatic H), 7.00 (d,J=9.1 Hz, 1H, aromatic H), 2.58-2.51 (m, 1H, cyclohexyl H), 2.01-1.64(m, 5H, cyclohexyl H), 1.56-1.10 (m, 5H, cyclohexyl H). ¹³C NMR (101MHz, CDCl₃) δ 167.18, 150.71, 145.88, 145.17, 140.22, 134.89, 128.30,121.12, 111.30, 44.08, 34.59, 26.95, 26.19.

5-nitro-2-(4-(tert-pentyl)phenoxy)pyridine, I3-nB

Following procedure A, 2-chloro-5-nitropyridine (303 mg, 1.91 mmol, 1.00eq) and 4-tert-pentylphenol (397 mg, 2.42 mmol, 1.27 eq) were dissolvedin DMSO (6 mL). Anhydrous K₂CO₃ (403 mg, 2.92 mmol, 1.53 eq) was addedand the reaction mixture was stirred at room temperature for 7 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:100) to afford the titlecompound as colourless solid (530 mg, 1.85 mmol, 97% yield). R_(f)=0.31(EtOAc/PE 1:20). HRMS (ESI) calcd. for C₁₆H₁₉N₂O₃ ⁺ [M+H]⁺ 287.1390.found: 287.1381. ¹H NMR (400 MHz, CDCl₃) δ 9.07-9.05 (m, 1H, aromaticH), 8.45 (dd, J=9.4, 2.4 Hz, 1H, aromatic H), 7.40 (d, J=8.6 Hz, 2H,aromatic H), 7.09 (d, J=8.6 Hz, 2H, aromatic H), 6.99 (d, J=9.2 Hz, 1H,aromatic H), 1.67 (q, J=7.4 Hz, 2H, Ar—C(CH₃)₂CH₂CH₃), 1.31 (s, 6H,Ar—C(CH₃)₂CH₂CH₃), 0.73 (t, J=7.4 Hz, 3H, Ar—C(CH₃)₂CH₂CH₃). ¹³C NMR(101 MHz, CDCl₃) δ 167.17, 150.46, 147.34, 145.20, 140.27, 134.90,127.56, 120.72, 111.28, 37.89, 37.06, 28.55, 9.26.

1-(tert-butyl)-4-(4-nitrophenoxy)benzene, I3-nC

Following procedure A, 4-fluoronitrobenzene (500 mg, 3.54 mmol, 1.00 eq)and 4-tert-butylphenol (671 mg, 4.46 mmol, 1.26 eq) were dissolved inDMF (6.0 mL). Anhydrous K₂CO₃ (857 mg, 6.20 mmol, 1.75 eq) was added andthe reaction mixture was stirred at room temperature for 52 h. Afterextraction with EtOAc, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:100) to afford the titlecompound as pale yellow solid (860 mg, 3.17 mmol, 89% yield). R_(f)=0.72(EtOAc/PE 1:9). HRMS (ESI) calcd. for C₁₆H₁₈NO₃ ⁺ [M+H]⁺ 272.1281.found: 272.1272. ¹H NMR (400 MHz, CDCl₃) δ 8.24-8.16 (m, 2H, aromaticH), 7.49-7.39 (m, 2H, aromatic H), 7.06-6.97 (m, 4H, aromatic H), 1.35(s, 9H, C(CH₃)₃). ¹³C NMR (101 MHz, CDCl₃) δ 163.82, 152.29, 148.59,142.54, 127.27, 126.03, 120.15, 116.98, 34.67, 31.58.

2-(4-butylphenoxy)-5-nitropyridine, I3-nD

Following procedure A, 2-chloro-5-nitropyridine (103 mg, 0.65 mmol, 1.00eq) and 4-butylphenol (126 mg, 0.84 mmol, 1.29 eq) were dissolved in DMF(2.5 mL). Anhydrous K₂CO₃ (143 mg, 1.04 mmol, 1.59 eq) was added and thereaction mixture was stirred at room temperature for 6 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:100) to afford the titlecompound as colourless solid (190 mg, 0.70 mmol, quant. yield).R_(f)=0.33 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C₁₅H₁₇N₂O₃ ⁺ [M+H]⁺273.1234. found: 273.1226. ¹H NMR (400 MHz, CDCl₃) δ 9.05 (dd, J=2.9,0.6 Hz, 1H, aromatic H), 8.46 (dd, J=9.1, 2.8 Hz, 1H, aromatic H),7.32-7.21 (m, 2H, aromatic H), 7.11-7.02 (m, 2H, aromatic H), 7.00 (dd,J=9.0, 0.6 Hz, 1H, aromatic H), 2.69-2.60 (m, 2H, Ar—CH₂CH₂CH₂CH₃),1.70-1.57 (m, 2H, Ar—CH₂CH₂CH₂CH₃), 1.39 (dq, J=14.6, 7.3 Hz, 2H,Ar—CH₂CH₂CH₂CH₃), 0.95 (t, J=7.3 Hz, 3H, Ar—CH₂CH₂CH₂CH₃). ¹³C NMR (101MHz, CDCl₃) δ 167.27, 150.75, 145.22, 140.86, 140.30, 134.92, 129.91,121.22, 111.32, 35.21, 33.67, 22.51, 14.08.

1-nitro-4-(4-(tert-pentyl)phenoxy)benzene, I3-nE

Following procedure A, 4-fluoronitrobenzene (318 mg, 2.25 mmol, 1.00 eq)and 4-tert-pentylphenol (460 mg, 2.28 mmol, 1.24 eq) were dissolved inDMSO (6 mL) Anhydrous K₂CO₃ (465 mg, 3.37 mmol, 1.49 eq) was added andthe reaction mixture was stirred at room temperature for 2 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:100) to afford the titlecompound as colourless solid (533 mg, 1.87 mmol, 83% yield). R_(f)=0.70(EtOAc/PE 1:20). HRMS (ESI) calcd. for C₁₇H₂₀NO₃ ⁺ [M+H]⁺ 285.1365.found: 285.1359. ¹H NMR (400 MHz, CDCl₃) δ 8.25-8.14 (m, 2H, aromaticH), 7.44-7.32 (m, 2H, aromatic H), 7.06-6.96 (m, 4H, aromatic H), 1.66(q, J=7.4 Hz, 2H, Ar—C(CH₃)₂CH₂CH₃), 1.31 (s, 6H, Ar—C(CH₃)₂CH₂CH₃),0.71 (t, J=7.4 Hz, 3H, Ar—C(CH₃)₂CH₂CH₃). ¹³C NMR (101 MHz, CDCl₃) δ163.81, 152.24, 146.94, 142.56, 127.91, 126.01, 120.06, 116.99, 37.89,37.06, 28.63, 9.27.

1-cyclohexyl-4-(4-nitrophenoxy)benzene, I3-nF

Following procedure A, 4-fluoronitrobenzene (325 mg, 2.30 mmol, 1.00 eq)and 4-cyclohexylphenol (519 mg, 2.94 mmol, 1.28 eq) were dissolved inDMSO (6 mL). Anhydrous K₂CO₃ (513 mg, 3.72 mmol, 1.61 eq) was added andthe reaction mixture was stirred at room temperature for 48 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:100 to 1:50) to afford thetitle compound as pale yellow solid (640 mg, 2.15 mmol, 93% yield).R_(f)=0.55 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C₁₈H₂₀NO₃ ⁺ [M+H]⁺298.1438. found: 298.1442. ¹H NMR (400 MHz, CDCl₃) δ 8.31-8.11 (m, 2H,aromatic H), 7.26 (d, J=2.3 Hz, 2H, aromatic H), 7.13-6.92 (m, 4H,aromatic H), 2.57-2.50 (m, 1H, cyclohexyl H), 2.09-1.67 (m, 5H,cyclohexyl H), 1.59-1.18 (m, 5H, cyclohexyl H). ¹³C NMR (101 MHz, CDCl₃)δ 163.89, 152.61, 145.58, 142.57, 128.66, 126.04, 120.49, 116.99, 44.14,34.72, 26.99, 26.23.

2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)-5-nitropyridine, I3-nG

Following procedure A, 2-chloro-5-nitropyridine (300 mg, 1.89 mmol, 1.00eq) and 4-adamantylphenol (546 mg, 2.39 mmol, 1.26 eq) were dissolved inDMSO (6 mL) Anhydrous K₂CO₃ (662 mg, 4.79 mmol, 2.53 eq) was added andthe reaction mixture was stirred at room temperature for 42 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:100 to 1:50) to afford thetitle compound as colourless solid (664 mg, 1.89 mmol, quant. yield).R_(f)=0.36 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C₂₁H₂₃N₂O₃ ⁺ [M+H]⁺351.1703. found: 351.1701. ¹H NMR (400 MHz, CDCl₃) δ 9.06 (d, J=2.8 Hz,1H, aromatic H), 8.45 (dd, J=9.1, 2.8 Hz, 1H, aromatic H), 7.52-7.39 (m,2H, aromatic H), 7.16-7.06 (m, 2H, aromatic H), 7.00 (d, J=9.1 Hz, 1H,aromatic H), 2.13-2.10 (m, 3H, adamantyl H), 1.94 (d, J=3.0 Hz, 5H,adamantyl H), 1.87-1.70 (m, 5H, adamantyl H). ¹³C NMR (101 MHz, CDCl₃) δ167.19, 150.51, 149.18, 145.20, 140.26, 134.89, 126.53, 120.83, 111.32,43.35, 36.82, 36.18, 29.02.

2-(3-(tert-butyl)phenoxy)-5-nitropyridine, I3-nH

Following procedure A, 2-chloro-5-nitropyridine (300 mg, 1.89 mmol, 1.00eq) and 3-tert-butylphenol (358 mg, 2.38 mmol, 1.26 eq) were dissolvedin DMSO (6 mL). Anhydrous K₂CO₃ (420 mg, 3.04 mmol, 1.60 eq) was addedand the reaction mixture was stirred at room temperature for 70 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:100) to afford the titlecompound as colourless solid (512 mg, 1.88 mmol, 99% yield). R_(f)=0.37(EtOAc/PE 1:20). HRMS (ESI) calcd. for C₁₅H₁₇N₂O₃ ⁺ [M+H]⁺ 273.1234.found: 273.1232. ¹H NMR (400 MHz, CDCl₃) δ 9.08-9.04 (m, 2H, aromaticH), 8.47 (dd, J=9.1, 2.8 Hz, 1H, aromatic H), 7.39 (t, J=7.9 Hz, 1H,aromatic H), 7.33 (ddd, J=7.9, 1.8, 1.2 Hz, 1H, aromatic H), 7.17 (t,J=2.1 Hz, 1H, aromatic H), 7.01 (dd, J=9.1, 0.5 Hz, 1H, aromatic H),6.98 (ddd, J=7.9, 2.4, 1.2 Hz, 1H, aromatic H), 1.34 (s, 9H, C(CH₃)₃).¹³C NMR (101 MHz, CDCl₃) δ 167.21, 153.88, 152.76, 145.26, 140.31,134.93, 129.50, 123.17, 118.62, 118.47, 111.28, 35.02, 31.36.

1-(4-(tert-butyl)phenoxy)-2-fluoro-4-nitrobenzene, I3-nI

Following procedure A, 3,4-difluoronitrobenzene (401 mg, 2.52 mmol, 1.00eq) and 4-tert-butylphenol (477 mg, 3.18 mmol, 1.26 eq) were dissolvedin DMSO (6 mL). Anhydrous K₂CO₃ (522 mg, 3.78 mmol, 1.50 eq) was addedand the reaction mixture was stirred at room temperature for 19 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:50) to afford the titlecompound as colourless oil (722 mg, 2.52 mmol, 99% yield). R_(f)=0.54(EtOAc/PE 1:20). HRMS (ESI) calcd. for C₁₆H₁₇FNO₃ ⁺ [M+H]⁺ 290.1187.found: 290.1194. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (dd, J=10.3, 2.7 Hz, 1H,aromatic H), 7.96 (ddd, J=9.1, 2.7, 1.5 Hz, 1H, aromatic H), 7.49-7.38(m, 2H, aromatic H), 7.09-6.99 (m, 2H, aromatic H), 6.96 (dd, J=9.1, 8.0Hz, 1H, aromatic H), 1.35 (s, 9H, C(CH₃)₃). ¹³C NMR (101 MHz, CDCl₃) δ153.35, 152.23, 151.93, 151.82, 150.84, 148.70, 142.40, 142.33, 127.29,120.68, 120.64, 119.38, 117.73, 117.71, 113.28, 113.05, 34.65, 31.54.

1-(4-cyclohexylphenoxy)-2-fluoro-4-nitrobenzene, I3-nJ

Following procedure A, 3,4-difluoronitrobenzene (509 mg, 3.20 mmol, 1.00eq) and 4-cyclohexylphenol (691 mg, 3.93 mmol, 1.23 eq) were dissolvedin DMSO (6 mL). Anhydrous K₂CO₃ (664 mg, 4.81 mmol, 1.50 eq) was addedand the reaction mixture was stirred at room temperature for 23 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:50) to afford the titlecompound as pale yellow solid (1002 mg, 3.18 mmol, 99% yield).R_(f)=0.51 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C₁₈H₁₉FNO₃ ⁺ [M+H]⁺316.1343. found: 316.1348. ¹H NMR (400 MHz, CDCl₃) δ 8.09 (dd, J=10.3,2.7 Hz, 1H, aromatic H), 7.97 (ddd, J=9.1, 2.7, 1.4 Hz, 1H, aromatic H),7.33-7.22 (m, 2H, aromatic H), 7.08-6.99 (m, 2H, aromatic H), 6.96 (dd,J=9.1, 8.0 Hz, 1H, aromatic H), 2.58-251 (m, 1H, cyclohexyl H),1.98-1.73 (m, 5H, cyclohexyl H), 1.52-1.20 (m, 5H, cyclohexyl H). ¹³CNMR (101 MHz, CDCl₃) δ 153.35, 152.51, 152.01, 151.90, 150.84, 145.68,142.39, 142.32, 128.67, 120.70, 120.66, 119.73, 117.70, 117.68, 113.30,113.08, 44.09, 34.69, 26.97, 26.21.

2-fluoro-4-nitro-1-(4-(tert-pentyl)phenoxy)benzene, I3-nK

Following procedure A, 3,4-difluoronitrobenzene (502 mg, 3.16 mmol, 1.00eq) and 4-tert-pentylphenol (693 mg, 4.22 mmol, 1.34 eq) were dissolvedin DMSO (6 mL) Anhydrous K₂CO₃ (656 mg, 4.75 mmol, 1.50 eq) was addedand the reaction mixture was stirred at room temperature for 22 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:50) to afford the titlecompound as pale yellow oil (943 mg, 3.11 mmol, 99% yield). R_(f)=0.61(EtOAc/PE 1:20). HRMS (ESI) calcd. for C₁₇H₁₉NO₃ ⁺ [M+H]⁺ 304.1343.found: 304.1332. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (dd, J=10.3, 2.7 Hz, 1H,aromatic H), 7.96 (ddd, J=9.1, 2.7, 1.5 Hz, 1H, aromatic H), 7.42-7.35(m, 2H, aromatic H), 7.07-6.99 (m, 2H, aromatic H), 6.95 (dd, J=9.1, 8.0Hz, 1H, aromatic H), 1.66 (q, J=7.4 Hz, 2H, Ar—C(CH₃)₂CH₂CH₃), 1.31 (s,6H, Ar—C(CH₃)₂CH₂CH₃), 0.71 (t, J=7.4 Hz, 3H, Ar—C(CH₃)₂CH₂CH₃). ¹³C NMR(101 MHz, CDCl₃) δ 153.40, 152.19, 151.98, 151.87, 150.88, 147.11,127.97, 120.71, 120.68, 119.34, 117.73, 117.71, 113.11, 37.92, 37.08,28.64.

2-(4-(2-methylpentan-2-yl)phenoxy)-5-nitropyridine, I3-nL

Following procedure A, 4-(2-methylpentan-2-yl)phenol (52 mg, 0.29 mmol,1.00 eq) and 2-chloro-5-nitropyridine (57 mg, 0.36 mmol, 1.23 eq) weredissolved in DMSO (6 mL). Anhydrous K₂CO₃ (66 mg, 0.48 mmol, 1.65 eq)was added and the reaction mixture was stirred at room temperature for27 h. After extraction with Et₂O, the crude product was purified byflash column chromatography (SiO₂; EtOAc/petrolether 1:50) to afford thetitle compound as colourless solid (86 mg, 0.29 mmol, 98% yield).R_(f)=0.50 (EtOAc/PE 1:10). HRMS (ESI) calcd. for C₁₇H₂₁N₂O₃ ⁺ [M+H]⁺301.1547. found: 301.1545. ¹H NMR (400 MHz, CDCl₃) δ 9.07 (d, J=2.7 Hz,1H, aromatic H), 8.46 (dd, J=9.1, 2.8 Hz, 1H, aromatic H), 7.45-7.35 (m,2H, aromatic H), 7.14-7.05 (m, 2H, aromatic H), 6.99 (dd, J=9.0, 0.6 Hz,1H, aromatic H), 1.65-1.54 (m, 2H, C(CH₃)₂CH₂CH₂CH₃), 1.32 (s, 6H,C(CH₃)₂CH₂CH₂CH₃), 1.19-1.05 (m, 2H, C(CH₃)₂CH₂CH₂CH₃), 0.84 (t, J=7.3Hz, 3H, C(CH₃)₂CH₂CH₂CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 167.17, 150.43,147.69, 145.21, 140.26, 134.90, 127.44, 120.72, 111.29, 47.27, 37.74,29.07, 18.08, 14.87.

(3r,5r,7r)-1-(4-(4-nitrophenoxy)phenyl)adamantane, I3-nM

Following procedure A, 4-fluoronitrobenzene (303 mg, 2.15 mmol, 1.00 eq)and 4-adamantylphenol (600 mg, 2.63 mmol, 1.22 eq) were dissolved inDMSO (6 mL) Anhydrous K₂CO₃ (450 mg, 3.26 mmol, 1.52 eq) was added andthe reaction mixture was stirred at room temperature for 20 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:100 to 1:50) to afford thetitle compound as pale yellow solid (736 mg, 2.11 mmol, 98% yield).R_(f)=0.73 (EtOAc/PE 1:10). HRMS (ESI) calcd. for C₂₂H₂₄NO₃ ⁺ [M+H]⁺350.1751. found: 350.1760. ¹H NMR (400 MHz, CDCl₃) δ 8.27-8.10 (m, 2H,aromatic H), 7.46-7.35 (m, 2H, aromatic H), 7.08-6.95 (m, 4H, aromaticH), 2.13-2.10 (m, 3H, adamantyl H), 1.93 (d, J=2.9 Hz, 6H, adamantyl H),1.88-1.70 (m, 6H, adamantyl H). ¹³C NMR (101 MHz, CDCl₃) δ 163.85,152.30, 148.86, 142.53, 126.86, 126.85, 126.03, 120.18, 117.00, 43.40,36.82, 36.17, 29.03.

(3r,5r,7r)-1-(4-(2-fluoro-4-nitrophenoxy)phenyl)adamantane, I3-nN

Following procedure A, 3,4-difluoronitrobenzene (303 mg, 1.90 mmol, 1.00eq) and 4-adamantylphenol (533 mg, 2.34 mmol, 1.23 eq) were dissolved inDMSO (6 mL) Anhydrous K₂CO₃ (395 mg, 2.86 mmol, 1.50 eq) was added andthe reaction mixture was stirred at room temperature for 4 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:100) to afford the titlecompound as colourless solid (703 mg, 1.91 mmol, quant. yield).R_(f)=0.69 (EtOAc/PE 1:10). HRMS (APPI) calcd. for C₂₂H₂₂FNO⁺ [M]⁺367.1584. found: 367.1581. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (dd, J=10.3,2.7 Hz, 1H, aromatic H), 7.96 (ddd, J=9.1, 2.7, 1.5 Hz, 1H, aromatic H),7.46-7.36 (m, 2H, aromatic H), 7.06-7.00 (m, 2H, aromatic H), 6.95 (dd,J=9.1, 8.0 Hz, 1H, aromatic H), 2.17-2.07 (m, 3H, adamantyl H), 1.92 (d,J=2.9 Hz, 5H, adamantyl H), 1.87-1.71 (m, 5H, adamantyl H). ¹³C NMR (101MHz, CDCl₃) δ 153.36, 153.35, 152.24, 152.21, 151.99, 151.88, 150.85,150.83, 148.98, 126.89, 120.70, 120.66, 119.44, 119.42, 117.72, 117.70,113.30, 113.07, 43.38, 36.81, 36.17, 29.02.

2-(4-isopropylphenoxy)-5-nitropyridine, I3-n0

Following procedure A, 2-chloro-5-nitropyridine (303 mg, 1.91 mmol, 1.00eq) and 4-iso-propylphenol (331 mg, 2.43 mmol, 1.27 eq) were dissolvedin DMF (6.0 mL). Anhydrous K₂CO₃ (398 mg, 2.88 mmol, 1.51 eq) was addedand the reaction mixture was stirred at room temperature for 24 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:100) to afford the titlecompound as pale yellow solid (490 mg, 1.90 mmol, 99% yield). R_(f)=0.40(EtOAc/PE 1:20). HRMS (ESI) calcd. for C₁₄H₁₅N₂O₃ ⁺ [M+H]⁺ 259.1077.found: 259.1072. ¹H NMR (400 MHz, CDCl₃) δ 9.04 (d, J=3.4 Hz, 1H,aromatic H), 8.44 (dd, J=9.1, 2.8 Hz, 1H, aromatic H), 7.37-7.28 (m, 2H,aromatic H), 7.12-7.06 (m, 2H, aromatic H), 7.03-6.97 (m, 1H, aromaticH), 2.96 (hept, J=6.9 Hz, 1H, CH(CH₃)₂), 1.30 (d, J=7.0 Hz, 6H,CH(CH₃)₂). ¹³C NMR (101 MHz, CDCl₃) δ 167.08, 150.67, 146.49, 145.02,140.18, 134.81, 127.84, 121.11, 111.24, 33.62, 24.03.

5-nitro-2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)pyridine, I3-nP

Following procedure A, 2-Chloro-5-nitropyridine (303 mg, 1.91 mmol, 1.00eq) and 4-tert-octylphenol (495 mg, 2.40 mmol, 1.25 eq) were dissolvedin DMSO (5 mL). Anhydrous K₂CO₃ (426 mg, 3.08 mmol, 1.61 eq) was addedand the reaction mixture was stirred at 40° C. for 28 h. Afterextraction with Et₂O, the crude product was purified by flash columnchromatography (SiO₂; EtOAc/petrolether 1:50) to afford the titlecompound as colourless solid (598 mg, 1.82 mmol, 95% yield). R_(f)=0.65(EtOAc/PE 1:10). HRMS (ESI) calcd. for C₁₉H₂₅N₂O₃ ⁺ [M+H]⁺ 329.1860.found: 329.1854. ¹H NMR (400 MHz, CDCl₃) δ 9.07 (d, J=2.8 Hz, 1H,aromatic H), 8.45 (dd, J=9.1, 2.8 Hz, 1H, aromatic H), 7.52-7.40 (m, 2H,aromatic H), 7.14-7.03 (m, 2H, aromatic H), 6.97 (d, J=9.1 Hz, 1H,aromatic H), 1.76 (s, 2H, Ar—C(CH₃)₂CH₂C(CH₃)₃), 1.40 (s, 6H,Ar—C(CH₃)₂CH₂C(CH₃)₃), 0.75 (s, 9H, Ar—C(CH₃)₂CH₂C(CH₃)₃). ¹³C NMR (101MHz, CDCl₃) δ 167.24, 150.49, 148.12, 145.30, 140.29, 134.92, 127.78,120.57, 111.15, 57.28, 38.63, 32.55, 31.93, 31.62.

General Procedure B:

The respective nitro derivatives (I3-n, I3-nA to I3-nP) were firstdissolved in MeOH or toluene, or directly added to a suspension ofcatalytic amounts of Pd (10%) on activated carbon powder in MeOH. Theflask was purged with H₂ (6×) and the reaction mixture was stirred atroom temperature until complete conversion. The reaction mixture wasthen filtered through Celite. The solvent was removed under reducedpressure at 30° C. and the crude product was purified by flash columnchromatography to give the corresponding title compounds (I3, I3-A toI3-P).

6-(4-(tert-butyl)phenoxy)pyridin-3-amine, I3

Following procedure B, I3-n (300 mg, 1.10 mmol, 1.00 eq) was added to asuspension of Pd (10%) on activated carbon powder (82 mg, 0.08 mmol Pd,0.07 eq) in MeOH (15 mL). The flask was purged with H₂ (6×) and thereaction mixture was stirred at room temperature for 2 h. The crudeproduct was purified by flash column chromatography (SiO2; DCM/MeOH 1%)to give the title compound as pale beige solid (250 mg, 1.03 mmol, 94%yield). R_(f)=0.40 (DCM/MeOH 4%). HRMS (ESI) calcd. for C₁₅H₁₉N₂O⁺[M+H]⁺ 243.1492. found: 243.1487. ¹H NMR (400 MHz, CDCl₃) δ 7.69 (d,J=3.0 Hz, 1H, aromatic H), 7.39-7.31 (m, 2H, aromatic H), 7.03 (dd,J=8.6, 3.0 Hz, 1H, aromatic H), 7.00-6.93 (m, 2H, aromatic H), 6.72 (d,J=8.6 Hz, 1H, aromatic H), 3.48 (s, 1H, NH₂), 1.31 (s, 9H, C(CH₃)₃). ¹³CNMR (101 MHz, CDCl₃) δ 156.62, 153.28, 146.33, 138.82, 134.06, 126.86,126.47, 119.24, 112.36, 34.33, 31.52.

6-(4-cyclohexylphenoxy)pyridin-3-amine, I3-A

Following procedure B, I3-nA (201 mg, 0.67 mmol, 1.00 eq) was added to asuspension of Pd (10%) on activated carbon powder (47 mg, 0.04 mmol Pd,0.07 eq) in MeOH (15 mL). The flask was purged with H₂ (6×) and thereaction mixture was stirred at room temperature for 5 h. The crudeproduct was purified by flash column chromatography (SiO₂; DCM/MeOH 1%)to give the title compound as beige solid (190 mg, 0.71 mmol, quant.yield). R_(f)=0.36 (DCM/MeOH 4%). HRMS (ESI) calcd. for C₁₇H₂₁N₂O⁺[M+H]⁺ 269.1648. found: 269.1643. ¹H NMR (400 MHz, CDCl₃) δ 7.70 (d,J=2.9 Hz, 1H, aromatic H), 7.22-7.13 (m, 2H, aromatic H), 7.04 (dd,J=8.6, 3.0 Hz, 1H, aromatic H), 7.01-6.92 (m, 2H, aromatic H), 6.73 (dd,J=8.8, 0.7 Hz, 1H, aromatic H), 3.36 (s, 2H, NH₂), 2.51-2.44 (m, 1H,cyclohexyl H), 1.97-1.67 (m, 5H, cyclohexyl H), 1.49-1.15 (m, 5H,cyclohexyl H). ¹³C NMR (101 MHz, CDCl₃) δ 156.88, 153.61, 143.47,138.67, 134.21, 127.92, 126.96, 119.68, 112.38, 43.99, 34.68, 27.01,26.25.

6-(4-(tert-pentyl)phenoxy)pyridin-3-amine, I3-B

Following procedure B, I3-nB (200 mg, 0.70 mmol, 1.00 eq) was added to asuspension of Pd (10%) on activated carbon powder (52 mg, 0.05 mmol Pd,0.07 eq) in MeOH (15 mL). The flask was purged with H₂ (6×) and thereaction mixture was stirred at room temperature for 3 h. The crudeproduct was purified by flash column chromatography (SiO₂; DCM/MeOH0.5%) to give the title compound as beige solid (107 mg, 0.42 mmol, 60%yield). R_(f)=0.41 (DCM/MeOH 4%). HRMS (ESI) calcd. for C₁₆H₂₁N₂O⁺[M+H]⁺ 257.1648. found: 257.1648. ¹H NMR (400 MHz, CDCl₃) δ 7.73 (d,J=2.9 Hz, 1H, aromatic H), 7.34-7.23 (m, 2H, aromatic H), 7.06 (dd,J=8.6, 3.0 Hz, 1H, aromatic H), 7.02-6.94 (m, 2H, aromatic H), 6.73 (d,J=8.6 Hz, 1H, aromatic H), 3.35 (s, 2H, NH₂), 1.62 (q, J=7.4 Hz, 2H,Ar—C(CH₃)₂CH₂CH₃), 1.27 (s, 6H, Ar—C(CH₃)₂CH₂CH₃), 0.70 (t, J=7.4 Hz,3H, Ar—C(CH₃)₂CH₂CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 156.80, 153.36,144.75, 138.72, 134.30, 127.19, 126.97, 119.13, 112.51, 37.63, 37.04,28.64, 9.26.

4-(4-(tert-butyl)phenoxy)aniline, I3-C

Following procedure B, I3-nC (300 mg, 1.11 mmol, 1.00 eq) was added to asuspension of Pd (10%) on activated carbon powder (59 mg, 0.06 mmol Pd,0.05 eq) in MeOH (15 mL). The flask was purged with H₂ (6×) and thereaction mixture was stirred at room temperature for 3.5 h. The crudeproduct was purified by flash column chromatography (SiO₂; ethylacetate/petrolether 1:100 to 1:10) to give the title compound as darkyellow oil (244 mg, 1.01 mmol, 91% yield). R_(f)=0.78 (DCM/MeOH 4%).HRMS (ESI) calcd. for C₁₆H₂₀NO⁺ [M+H]⁺ 242.1539. found: 242.1530. ¹H NMR(400 MHz, CDCl₃) δ 7.33-7.27 (m, 2H, aromatic H), 6.91-6.84 (m, 4H,aromatic H), 6.72-6.66 (m, 2H, aromatic H), 3.49 (s, 2H, NH₂), 1.31 (s,9H, C(CH₃)₃). ¹³C NMR (101 MHz, CDCl₃) δ 156.55, 149.24, 145.05, 142.30,126.45, 121.08, 116.90, 116.49, 34.33, 31.66.

6-(4-butylphenoxy)pyridin-3-amine, I3-D

Following procedure B, I3-nD (170 mg, 0.62 mmol, 1.00 eq) was added to asuspension of Pd (10%) on activated carbon powder (53 mg, 0.05 mmol Pd,0.08 eq) in MeOH (15 mL). The flask was purged with H₂ (6×) and thereaction mixture was stirred at room temperature for 1.5 h. The crudeproduct was purified by flash column chromatography (SiO₂; DCM/MeOH 1%)to give the title compound as brown oil (133 mg, 0.55 mmol, 88% yield).R_(f)=0.38 (DCM/MeOH 4%). HRMS (ESI) calcd. for C₁₅H₁₉N₂O⁺ [M+H]⁺243.1492. found: 243.1485. ¹H NMR (400 MHz, CDCl₃) δ 7.69 (d, J=3.1 Hz,1H, aromatic H), 7.20-7.09 (m, 2H, aromatic H), 7.03 (dd, J=8.6, 3.0 Hz,1H, aromatic H), 7.00-6.91 (m, 2H, aromatic H), 6.72 (d, J=8.6 Hz, 1H,aromatic H), 3.36 (s, 2H, NH₂), 2.67-2.51 (m, 2H, Ar—CH₂CH₂CH₂CH₃),1.66-1.52 (m, 2H, Ar—CH₂CH₂CH₂CH₃), 1.41-1.31 (m, 2H, Ar—CH₂CH₂CH₂CH₃),0.93 (t, J=7.3 Hz, 3H, Ar—CH₂CH₂CH₂CH₃). ¹³C NMR (101 MHz, CDCl₃) δ156.83, 153.55, 138.75, 138.26, 134.12, 129.48, 126.90, 119.74, 112.28,35.02, 33.74, 22.40, 14.02.

4-(4-(tert-pentyl)phenoxy)aniline, I3-E

Following procedure B, I3-nE (313 mg, 1.10 mmol, 1.00 eq) was dissolvedin MeOH (10 mL) and added to a suspension of Pd (10%) on activatedcarbon powder (52 mg, 0.05 mmol Pd, 0.04 eq) in MeOH (5 mL). The flaskwas purged with H₂ (6×) and the reaction mixture was stirred at roomtemperature for 3 h. The crude product was purified by filtrationthrough a thin SiO₂ layer (DCM/MeOH 4%) to give the title compound asbeige oil (293 mg, 1.15 mmol, quant. yield). R_(f)=0.09 (EtOAc/PE 1:20).HRMS (ESI) calcd. for C₁₇H₂₂N₂O⁺ [M+H]⁺ 256.1696. found: 256.1692. ¹HNMR (400 MHz, CDCl₃) δ 7.29-7.19 (m, 2H, aromatic H), 6.98-6.81 (m, 4H,aromatic H), 6.76-6.61 (m, 2H, aromatic H), 3.47 (s, 2H, NH₂), 1.63 (q,J=7.4 Hz, 2H, Ar—C(CH₃)₂CH₂CH₃), 1.28 (s, 6H, Ar—C(CH₃)₂CH₂CH₃), 0.71(t, J=7.4 Hz, 3H, Ar—C(CH₃)₂CH₂CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 156.45,149.08, 143.29, 142.55, 127.08, 121.04, 116.79, 116.33, 37.50, 37.06,28.69, 9.27.

4-(4-cyclohexylphenoxy)aniline, I3-F

Following procedure B, I3-nF (352 mg, 1.18 mmol, 1.00 eq) was dissolvedin toluene (5 mL) and added to a suspension of Pd (10%) on activatedcarbon powder (38 mg, 0.04 mmol Pd, 0.03 eq) in MeOH (10 mL). The flaskwas purged with H₂ (6×) and the reaction mixture was stirred at roomtemperature for 2 h. The crude product was purified by flash columnchromatography (SiO₂; DCM) to give the title compound as beige solid(315 mg, 1.18 mmol, quant. yield). R_(f)=0.86 (DCM/MeOH 4%). HRMS (ESI)calcd. for C₁₈H₂₂NO⁺ [M+H]⁺ 268.1696. found: 268.1692. ¹H NMR (400 MHz,CDCl₃) δ 7.17-7.07 (m, 2H, aromatic H), 6.94-6.82 (m, 4H, aromatic H),6.72-6.62 (m, 2H, aromatic H), 3.49 (s, 2H, NH₂), 2.51-2.44 (m, 1H,cyclohexyl H), 1.98-1.68 (m, 5H, cyclohexyl H), 1.46-1.21 (m, 5H,cyclohexyl H). ¹³C NMR (101 MHz, CDCl₃) δ 156.87, 149.13, 142.53,142.05, 127.81, 121.02, 117.22, 116.33, 43.90, 34.79, 27.05, 26.27.

6-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)pyridin-3-amine, I3-G

Following procedure B, I3-nG (278 mg, 0.79 mmol, 1.00 eq) was dissolvedin toluene (10 mL) and added to a suspension of Pd (10%) on activatedcarbon powder (56 mg, 0.05 mmol Pd, 0.07 eq) in MeOH (10 mL). The flaskwas purged with H₂ (3×) and the reaction mixture was stirred at roomtemperature for 3 h. The crude product was purified by flash columnchromatography (SiO₂; DCM/MeOH 0% to 1%) to give the title compound ascolourless solid (176 mg, 0.55 mmol, 69% yield). R_(f)=0.43 (DCM/MeOH4%). HRMS (ESI) calcd. for C₂₁H₂₅N₂O⁺ [M+H]⁺ 321.1961. found: 321.1959.¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J=3.0 Hz, 1H, aromatic H), 7.37-7.29(m, 2H, aromatic H), 7.08-6.96 (m, 3H, aromatic H), 6.74 (d, J=8.6 Hz,1H, aromatic H), 3.40 (s, 2H, NH₂), 2.12-2.09 (m, 3H, adamantyl H), 1.93(dd, J=9.7, 3.0 Hz, 5H, adamantyl H), 1.86-1.70 (m, 5H, adamantyl H).¹³C NMR (101 MHz, CDCl₃) δ 156.73, 153.37, 151.35, 146.65, 138.76,134.15, 128.14, 126.86, 126.06, 125.54, 124.88, 119.29, 112.40, 43.36,43.22, 36.87, 36.84, 35.87, 29.02.

6-(3-(tert-butyl)phenoxy)pyridin-3-amine, I3-H

Following procedure B, I3-nH (362 mg, 1.33 mmol, 1.00 eq) was dissolvedin MeOH (10 mL) and added to a suspension of Pd (10%) on activatedcarbon powder (44 mg, 0.04 mmol Pd, 0.03 eq) in MeOH (5 mL). The flaskwas purged with H₂ (5×) and the reaction mixture was stirred at roomtemperature for 2 h. The crude product was purified by flash columnchromatography (SiO₂; DCM/MeOH 0% to 1%) to give the title compound aspale brown oil (303 mg, 1.25 mmol, 94% yield). R_(f)=0.44 (DCM/MeOH 4%).HRMS (ESI) calcd. for C₁₅H₁₉N₂O⁺ [M+H]⁺ 243.1492. found: 243.1493. ¹HNMR (400 MHz, CDCl₃) δ 7.92 (d, J=3.0 Hz, 1H, aromatic H), 7.48-7.40 (m,1H, aromatic H), 7.32 (ddd, J=7.8, 1.9, 1.0 Hz, 1H, aromatic H),7.29-7.27 (m, 1H, aromatic H), 7.24 (d, J=3.0 Hz, 1H, aromatic H), 7.01(ddd, J=8.0, 2.4, 1.0 Hz, 1H, aromatic H), 6.92 (dd, J=8.6, 0.7 Hz, 1H,aromatic H), 3.40 (s, 2H, NH₂) 1.48 (s, 9H, C(CH₃)₃). ¹³C NMR (101 MHz,CDCl₃) δ 156.81, 155.65, 153.31, 138.69, 134.40, 129.10, 126.99, 120.85,117.23, 116.69, 112.54, 34.89, 31.41.

4-(4-(tert-butyl)phenoxy)-3-fluoroaniline, I3-I

Following procedure B, I3-nI (501 mg, 1.73 mmol, 1.00 eq) was dissolvedin MeOH (13 mL) and added to a suspension of Pd (10%) on activatedcarbon powder (56 mg, 0.05 mmol Pd, 0.03 eq) in MeOH (2 mL). The flaskwas purged with H₂ (5×) and the reaction mixture was stirred at roomtemperature for 1.5 h. The crude product was purified by flash columnchromatography (SiO₂; DCM) to give the title compound as colourlesssolid (455 mg, 1.75 mmol, quant. yield). R_(f)=0.76 (DCM/MeOH 1%). HRMS(ESI) calcd. for C₁₆H₁₉FNO⁺ [M+H]⁺ 260.1445. found: 260.1442. ¹H NMR(400 MHz, CDCl₃) δ 7.37-7.29 (m, 2H, aromatic H), 6.94 (t, J=8.8 Hz, 1H,aromatic H), 6.91-6.85 (m, 2H, aromatic H), 6.52 (dd, J=12.0, 2.7 Hz,1H, aromatic H), 6.42 (ddd, J=8.6, 2.7, 1.3 Hz, 1H, aromatic H), 3.61(s, 2H, NH₂), 1.33 (s, 9H, C(CH₃)₃). ¹³C NMR (101 MHz, CDCl₃) δ 156.58,156.44, 154.12, 145.02, 144.42, 144.33, 134.83, 134.71, 126.41, 123.92,123.90, 115.43, 110.97, 110.93, 103.93, 103.72, 34.25, 31.59.

4-(4-cyclohexylphenoxy)-3-fluoroaniline, I3-J

Following procedure B, I3-nJ (550 mg, 1.74 mmol, 1.00 eq) was dissolvedin MeOH (12 mL) and added to a suspension of Pd (10%) on activatedcarbon powder (46 mg, 0.04 mmol Pd, 0.03 eq) in MeOH (3 mL). The flaskwas purged with H₂ (6×) and the reaction mixture was stirred at roomtemperature for 2 h. The crude product was purified by flash columnchromatography (SiO₂; DCM/petrolether 1:1 to 2:1) to give the titlecompound as pale rose solid (489 mg, 1.71 mmol, 98% yield). R_(f)=0.81(DCM/MeOH 4%). HRMS (ESI) calcd. for C₁₈H₂₁FNO⁺ [M+H]⁺ 286.1602. found:286.1613. ¹H NMR (400 MHz, CDCl₃) δ 7.19 ? 7.10 (m, 2H, aromatic H),6.93 (t, J=8.8 Hz, 1H, aromatic H), 6.90-6.83 (m, 2H, aromatic H), 6.51(dd, J=12.1, 2.7 Hz, 1H, aromatic H), 6.41 (ddd, J=8.6, 2.7, 1.2 Hz, 1H,aromatic H), 3.66 (s, 2H, NH₂), 2.52-2.45 (m, 1H, cyclohexyl H),1.96-1.72 (m, 5H, cyclohexyl H), 1.53-1.18 (m, 5H, cyclohexyl H). ¹³CNMR (101 MHz, CDCl₃) δ 156.74, 156.55, 154.09, 144.39, 144.30, 142.04,134.86, 134.74, 127.79, 123.89, 115.76, 110.93, 110.90, 103.90, 103.69,43.80, 34.72, 26.99, 26.22.

3-fluoro-4-(4-(tert-pentyl)phenoxy)aniline, I3-K

Following procedure B, I3-nK (497 mg, 1.64 mmol, 1.00 eq) was dissolvedin MeOH (13 mL) and added to a suspension of Pd (10%) on activatedcarbon powder (28 mg, 0.03 mmol Pd, 0.02 eq) in MeOH (2 mL). The flaskwas purged with H₂ (6×) and the reaction mixture was stirred at roomtemperature for 1.5 h. The crude product was purified by flash columnchromatography (SiO₂; ethyl acetate/petrolether 1:10 to 1:7.5) to givethe title compound as orange oil (463 mg, 1.69 mmol, quant. yield).R_(f)=0.28 (EtOAc/petrolether 1:5). HRMS (ESI) calcd. for C₁₇H₂₁FNO⁺[M+H]⁺ 274.1602. found: 274.1599. ¹H NMR (400 MHz, CDCl₃) δ 7.26-7.17(m, 2H, aromatic H), 6.92 (t, J=8.8 Hz, 1H, aromatic H), 6.89-6.80 (m,2H, aromatic H), 6.51 (dd, J=12.0, 2.7 Hz, 1H, aromatic H), 6.42 (ddd,J=8.7, 2.7, 1.2 Hz, 1H, aromatic H), 3.65 (s, 2H, NH₂), 1.61 (q, J=7.4Hz, 2H, Ar—C(CH₃)₂CH₂CH₃), 1.26 (s, 6H, Ar—C(CH₃)₂CH₂CH₃), 0.69 (t,J=7.4 Hz, 3H, Ar—C(CH₃)₂CH₂CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 156.61,156.37, 154.15, 144.34, 144.25, 143.34, 134.98, 134.86, 123.95, 123.92,115.41, 110.98, 110.95, 104.00, 103.78, 37.07, 28.68, 9.26.

6-(4-(2-methylpentan-2-yl)phenoxy)pyridin-3-amine, I3-L

Following procedure B, I3-nL (50 mg, 0.17 mmol, 1.00 eq) was dissolvedin MeOH (12 mL) and added to a suspension of Pd (10%) on activatedcarbon powder (31 mg, 0.03 mmol Pd, 0.17 eq) in MeOH (3 mL). The flaskwas purged with H₂ (6×) and the reaction mixture was stirred at roomtemperature for 1.5 h. The crude product was purified by flash columnchromatography (SiO₂; DCM/MeOH 1%) to give the title compound as paleorange oil (39 mg, 0.14 mmol, 87% yield). R_(f)=0.41 (DCM/MeOH 4%). HRMS(ESI) calcd. for C₁₇H₂₃N₂O⁺ [M+H]⁺ 271.1805. found: 271.1796. ¹H NMR(400 MHz, CDCl₃) δ 7.73 (d, J=2.9 Hz, 1H, aromatic H), 7.32-7.26 (m, 2H,aromatic 1/), 7.07 (dd, J=8.6, 3.0 Hz, 1H, aromatic H), 7.01-6.94 (m,2H, aromatic H), 6.74 (d, J=8.6 Hz, 1H, aromatic H), 3.35 (s, 2H, NH₂),1.61-1.51 (m, 2H, C(CH₃)₂CH₂CH₂CH₃), 1.27 (s, 6H, C(CH₃)₂CH₂CH₂CH₃),1.16-1.01 (m, 2H, C(CH₃)₂CH₂CH₂CH₃), 0.82 (t, J=7.3 Hz, 3H,C(CH₃)₂CH₂CH₂CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 156.82, 153.32, 145.10,138.67, 134.32, 128.12, 127.09, 126.99, 119.62, 119.13, 112.53, 47.31,37.49, 29.17, 18.08, 14.90.

4-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)aniline, I3-M

Following procedure B, I3-nM (615 mg, 1.76 mmol, 1.00 eq) was dissolvedin toluene (15 mL) and added to a suspension of Pd (10%) on activatedcarbon powder (126 mg, 0.12 mmol Pd, 0.07 eq) in MeOH (10 mL). The flaskwas purged with H₂ (6×) and the reaction mixture was stirred at roomtemperature for 19 h. The crude product was purified by flash columnchromatography (SiO₂; DCM) to give the title compound as beige solid(538 mg, 1.68 mmol, 96% yield). R_(f)=0.39 (DCM/MeOH 1%). HRMS (ESI)calcd. for C₂₂H₂₆NO⁺ [M+H]⁺ 320.2009. found: 320.2006. ¹H NMR (400 MHz,CDCl₃) δ 7.32-7.25 (m, 2H, aromatic H), 6.93-6.86 (m, 4H, aromatic H),6.72-6.65 (m, 2H, aromatic H), 3.56 (s, 2H, NH₂), 2.14-2.06 (m, 3H,adamantyl H), 1.90 (d, J=2.9 Hz, 5H, adamantyl H), 1.84-1.71 (m, 5H,adamantyl H). ¹³C NMR (101 MHz, CDCl₃) δ 156.59, 149.02, 145.32, 142.51,125.98, 121.10, 116.84, 116.34, 43.45, 36.88, 35.78, 29.07.

4-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)-3-fluoroaniline, I3-N

Following procedure B, I3-nN (570 mg, 1.55 mmol, 1.00 eq) was dissolvedin toluene (10 mL) and added to a suspension of Pd (10%) on activatedcarbon powder (143 mg, 0.13 mmol Pd, 0.09 eq) in MeOH (10 mL). The flaskwas purged with H₂ (6×) and the reaction mixture was stirred at roomtemperature for 7 h. The crude product was purified by flash columnchromatography (SiO₂; DCM) to give the title compound as colourlesssolid (510 mg, 1.51 mmol, 97% yield). R_(f)=0.67 (DCM/MeOH 1%). HRMS(ESI) calcd. for C₂₂H₂₅FNO⁺ [M+H]⁺ 338.1915. found: 338.1916. ¹H NMR(400 MHz, CDCl₃) δ 7.32-7.23 (m, 2H, aromatic H), 6.92 (t, J=8.8 Hz, 1H,aromatic H), 6.89-6.81 (m, 2H, aromatic H), 6.51 (dd, J=12.0, 2.7 Hz,1H, aromatic H), 6.41 (ddd, J=8.7, 2.7, 1.2 Hz, 1H, aromatic H), 3.64(s, 2H, NH₂), 2.11-2.07 (m, 3H, adamantyl H), 1.89 (d, J=2.9 Hz, 5H,adamantyl H), 1.83-1.70 (m, 5H, adamantyl H). ¹³C NMR (101 MHz, CDCl₃) δ156.60, 156.47, 154.15, 145.37, 144.33, 144.24, 134.91, 134.79, 125.98,123.97, 123.95, 115.47, 110.98, 110.95, 103.98, 103.76, 43.43, 36.86,35.76, 29.06.

6-(4-isopropylphenoxy)pyridin-3-amine, I3-0

Following procedure B, I3-nO (202 mg, 0.78 mmol, 1.00 eq) was added to asuspension of Pd (10%) on activated carbon powder (76 mg, 0.07 mmol Pd,0.09 eq) in MeOH (15 mL). The flask was purged with H₂ (6×) and thereaction mixture was stirred at room temperature for 1 h. The crudeproduct was purified by flash column chromatography (SiO₂; DCM/MeOH 1%)to give the title compound as beige solid (150 mg, 0.66 mmol, 84%yield). R_(f)=0.42 (DCM/MeOH 4%). HRMS (ESI) calcd. for C₁₄H₁₇N₂O⁺[M+H]⁺ 229.1335. found: 229.1326. ¹H NMR (400 MHz, CDCl₃) δ 7.70 (d,J=2.9 Hz, 1H, aromatic H), 7.24-7.14 (m, 2H, aromatic H), 7.05 (dd,J=8.6, 3.0 Hz, 1H, aromatic H), 7.01-6.93 (m, 2H, aromatic H), 6.73 (d,J=8.6 Hz, 1H, aromatic H), 3.33 (s, 2H, NH₂), 2.89 (hept, J=6.9 Hz, 1H,CH(CH₃)₂), 1.24 (d, J=7.0 Hz, 6H, CH(CH₃)₂). ¹³C NMR (101 MHz, CDCl₃) δ156.88, 153.59, 144.19, 138.68, 134.19, 127.55, 126.97, 119.76, 112.35,77.48, 77.16, 76.84, 33.57, 24.20.

6-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)pyridin-3-amine, I3-P

Following procedure B, I3-nP (283 mg, 0.86 mmol, 1.00 eq) was dissolvedin toluene (4 mL) and added to a suspension of Pd (10%) on activatedcarbon powder (78 mg, 0.07 mmol Pd, 0.09 eq) in MeOH (15 mL). The flaskwas purged with H₂ (5×) and the reaction mixture was stirred at roomtemperature for 2 h. The crude product was purified by flash columnchromatography (SiO₂; DCM/MeOH 2%) to give the title compound ascolourless solid (235 mg, 0.79 mmol, 91% yield). R_(f)=0.49 (DCM/MeOH4%). HRMS (ESI) calcd. for C₁₉H₂₇N₂O⁺ [M+H]⁺ 299.2118. found: 299.2112.¹H NMR (400 MHz, CDCl₃) δ 7.73 (dd, J=3.0, 0.7 Hz, 1H, aromatic H),7.37-7.29 (m, 2H, aromatic H), 7.06 (dd, J=8.6, 3.0 Hz, 1H, aromatic H),6.99-6.93 (m, 2H, aromatic H), 6.71 (dd, J=8.6, 0.7 Hz, 1H, aromatic H),3.10 (s, 2H, NH₂), 1.72 (s, 2H, Ar—C(CH₃)₂CH₂C(CH₃)₃), 1.36 (s, 6H,Ar—C(CH₃)₂CH₂C(CH₃)₃), 0.73 (s, 9H, Ar—C(CH₃)₂CH₂C(CH₃)₃). ¹³C NMR (101MHz, CDCl₃) δ 156.87, 153.39, 145.48, 138.70, 134.39, 127.37, 126.94,118.96, 112.41, 57.20, 38.36, 32.50, 31.93, 31.68.

1. 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I

or one of its derivatives having Notch signalling pathway inhibitionproperties, salts, solvates, tautomers, isomers thereof for use in thetreatment and/or prevention of a cancer. 2.6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I, or one of itsderivatives having Notch signalling pathway inhibition properties, ofclaim 1 wherein the cancer is a Notch dependent cancer. 3.6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I, or one of itsderivatives having Notch signalling pathway inhibition properties, ofclaim 2 wherein the Notch dependent cancer is selected from the groupcomprising T cell-Acute lymphoblastic leukemia (T-ALL), chronic myeloidleukemia (CML), chronic lymphocytic leukemia (CLL), Mantle cell lymphoma(MCL), breast cancer, pancreatic cancer, prostate cancer, melanoma,brain tumors, tumor angiogenesis, and colorectal cancer. 4.6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I, or one of itsderivatives having Notch signalling pathway inhibition properties, ofclaim 2 or 3 wherein the Notch dependent cancer is resistant toγ-secretase inhibitor treatment. 5.6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I, or one of itsderivatives having Notch signalling pathway inhibition properties, ofany one of the preceding claims wherein the derivative having Notchsignalling pathway inhibition properties is selected from the groupscomprising

wherein m is an integer selected from 1 to 4; W is selected from H andhalogens; the halogen is selected from F—, Cl—, Br— or I—; R1, R2, R3,R4 are each independently selected from the group consisting in H,phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl,furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl,tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n is an integerindependently selected from 1 to 15; X is O, S, CR5R6, NR7, NHCOR8, orNHSO2R9; where R5, R6, R7, R8, R9 are each independently selected fromthe group consisting in selected from the group consisting in H, phenyl,2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, or(CH2)_(n)CH3, the subscript n is an integer independently selected from1 to 15; Y is N or CH; Z is H, NO₂, OH, NR10R11 where R10 and R11 areeach independently selected from the group consisting in H and(CH₂)_(n)CH₃, NHCOR12 where R12 is selected from the group consisting of(CH₂)_(n)CH₃, aromatic and heteroaromatics such as phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, COOR13 whereR13 is selected from the group consisting of H, (CH₂)_(n)CH₃, aromaticand heteroaromatics such as phenyl, naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, NHSO₂R14 where R14 is selectedfrom the group consisting of phenyl, 2-, 3- or 4, substituted phenyl,naphthyl, heteroaromatics such as pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is aninteger independently selected from 1 to 15;

wherein m is an integer selected from 1 to 4; W is selected from H andhalogens; the halogen is selected from F—, Cl—, Br— or I—; R4, R15 areeach independently selected from the group consisting in H, phenyl, 2-,3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,(CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n is an integerindependently selected from 1 to 15; X is O, S, CR5R6, NR7, NHCOR8 orNHSO₂R9; R5, R6 and R7 are each independently selected from the groupconsisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃; R8 and R9 are eachindependently selected from the group consisting in phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected fromthe group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is an integerindependently selected from 1 to 15; Y is N or CH; Z is H, NO₂, OH,NR10R11 where R¹⁰ and R¹¹ are each independently selected from the groupconsisting in H, (CH₂)_(n)CH₃, NHCOR¹² where R12 is (CH₂)_(n)CH₃,aromatic and heteroaromatics selected from the group comprising phenyl,naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,COOR13 where R13 is H, (CH₂)_(n)CH₃, aromatic and heteroaromaticsselected from the group comprising phenyl, naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, NHSO₂R14 where R14 is phenyl, 2-,3- or 4, substituted phenyl, naphthyl, heteroaromatics selected from thegroup comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃, the subscript n is an integerindependently selected from 1 to 15;

wherein m is an integer selected from 1 to 4; W is selected from H andhalogens; the halogen is selected from F—, Cl—, Br— or I—; R4 is H,phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl,furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl,tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n is an integerindependently selected from 1 to 15; X is O, S, CR5R6, NR7, NHCOR8 orNHSO₂R9; R5, R6 and R7 are each independently selected from the groupconsisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃; R8 and R9 are eachindependently selected from the group consisting in phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected fromthe group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is an integerindependently selected from 1 to 15; Y is N or CH; Z is H, NO₂, OH,NR10R11 where R¹⁰ and R¹¹ are each independently selected from the groupconsisting in H, (CH₂)_(n)CH₃, NHCOR¹² where R12 is (CH₂)_(n)CH₃,aromatic and heteroaromatics selected from the group comprising phenyl,naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,COOR13 where R13 is H, (CH₂)_(n)CH₃, aromatic and heteroaromaticsselected from the group comprising phenyl, naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, NHSO₂R14 where R14 is phenyl, 2-,3- or 4, substituted phenyl, naphthyl, heteroaromatics selected from thegroup comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃, the subscript n is an integerindependently selected from 1 to 15;

wherein m is an integer selected from 1 to 4; W is selected from H andhalogens; the halogen is selected from F—, Cl—, Br— or I—; R1, R2, R3,R4 are each independently selected from the group consisting in H,phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl,furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl,tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n is an integerindependently selected from 1 to 15; X is O, S, CR5R6, NR7, NHCOR8 orNHSO₂R9; R5, R6 and R7 are each independently selected from the groupconsisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃; R8 and R9 are eachindependently selected from the group consisting in phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected fromthe group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is an integerindependently selected from 1 to 15; Z is H, NO₂, OH, NR10R11 where R10and R11 are each independently selected from the group consisting in H,(CH₂)_(n)CH₃, NHCOR12 where R12 is (CH₂)_(n)CH₃, aromatic andheteroaromatics selected form the group comprising phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, COOR13 whereR13 is H, (CH₂)_(n)CH₃, aromatic and heteroaromatics selected from thegroup comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4,substituted phenyl, naphthyl, heteroaromatics selected from the groupcomprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, (CH₂)_(n)CH₃; the subscript n is an integer independentlyselected from 1 to 15;

wherein m is an integer selected from 1 to 4; W is selected from H andhalogens; the halogen is selected from F—, Cl—, Br— or I—; R4, R15 areeach independently selected from the group consisting in H, phenyl, 2-,3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,(CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n is an integerindependently selected from 0 to 15; X is O, S, CR5R6, NR7, NHCOR8 orNHSO₂R9; R5, R6 and R7 are each independently selected from the groupconsisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃; R8 and R9 are eachindependently selected from the group consisting in phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected fromthe group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is an integerindependently selected from 0 to 15; Z is H, NO₂, OH, NR10R11 where R10and R11 are each independently selected from the group consisting in H,(CH₂)_(n)CH₃, NHCOR12 where R12 is (CH₂)_(n)CH₃, aromatic andheteroaromatics selected form the group comprising phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, COOR13 whereR13 is H, (CH₂)_(n)CH₃, aromatic and heteroaromatics selected from thegroup comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4,substituted phenyl, naphthyl, heteroaromatics selected from the groupcomprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, (CH₂)_(n)CH₃; the subscript n is an integer independentlyselected from 0 to 15;

wherein m is an integer selected from 1 to 3; W is selected from H andhalogens; the halogen is selected from F—, Cl—, Br— or I—; R4 is H,phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl,furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl,tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n is an integerindependently selected from 0 to 15; X is O, S, CR5R6, NR7, NHCOR8 orNHSO₂R9; R5, R6 and R7 are each independently selected from the groupconsisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃; R8 and R9 are eachindependently selected from the group consisting in phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected fromthe group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is an integerindependently selected from 0 to 15; Z is H, NO₂, OH, NR10R11 where R10and R11 are each independently selected from the group consisting in H,(CH₂)_(n)CH₃, NHCOR12 where R12 is (CH₂)_(n)CH₃, aromatic andheteroaromatics selected form the group comprising phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, COOR13 whereR13 is H, (CH₂)_(n)CH₃, aromatic and heteroaromatics selected from thegroup comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4,substituted phenyl, naphthyl, heteroaromatics selected from the groupcomprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, (CH₂)_(n)CH₃; the subscript n is an integer independentlyselected from 0 to 15;

wherein the heteroaromatic is an aminopyrrole, aminofurane,aminothiofurane, or a pyrimidine; R1, R2, R3, R4 are each independentlyselected from the group consisting in H, phenyl, 2-, 3- or 4-substitutedphenyl, 2- or 3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl;the subscript n is an integer independently selected from 0 to 15; X isO, S, CR5R6, NR7, NHCOR8, or NHSO2R9; where R5, R6, R7, R8, R9 are eachindependently selected from the group consisting in H, phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, or (CH2)_(n)CH3,the subscript n is an integer independently selected from 0 to 15; Z isH, NO₂, OH, NR10R11 where R10 and R11 are each independently selectedfrom the group consisting in H, (CH₂)_(n)CH₃, NHCOR12 where R12 is(CH₂)_(n)CH₃, aromatic and heteroaromatics selected form the groupcomprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH₂)_(n)CH₃, aromaticand heteroaromatics selected from the group comprising phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, NHSO₂R14 withR14 is phenyl, 2-, 3- or 4, substituted phenyl, naphthyl,heteroaromatics selected from the group comprising pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH₂)_(n)CH₃; thesubscript n is an integer independently selected from 0 to 15;

wherein the subscript n is an integer independently selected from 1 to15; the heteroaromatic is an aminopyrrole, aminofurane, aminothiofurane,or a pyrimidine; R4, R15 are each independently selected from the groupconsisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; thesubscript n is an integer independently selected from 0 to 15; X is O,S, CR5R6, NR7, NHCOR8 or NHSO₂R9; R5, R6 and R7 are each independentlyselected from the group consisting in H, phenyl, 2-, 3- or 4-substitutedphenyl, 2- or 3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃; R8 and R9 areeach independently selected from the group consisting in phenyl, 2-, 3-or 4-substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selectedfrom the group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is an integerindependently selected from 0 to 15; Z is H, NO₂, OH, NR10R11 where R10and R11 are each independently selected from the group consisting in H,(CH₂)_(n)CH₃, NHCOR12 where R12 is (CH₂)_(n)CH₃, aromatic andheteroaromatics selected form the group comprising phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, COOR13 whereR13 is H, (CH₂)_(n)CH₃, aromatic and heteroaromatics selected from thegroup comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4,substituted phenyl, naphthyl, heteroaromatics selected from the groupcomprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, (CH₂)_(n)CH₃; the subscript n is an integer independentlyselected from 0 to 15;

wherein the heteroaromatic is an aminopyrrole, aminofurane,aminothiofurane, or a pyrimidine; R4 is H, phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃alkenyl, alkynyl; the subscript n is an integer independently selectedfrom 0 to 15; X is O, S, CR5R6, NR7, NHCOR8 or NHSO₂R9; R5, R6 and R7are each independently selected from the group consisting in H, phenyl,2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,(CH₂)_(n)CH₃; R8 and R9 are each independently selected from the groupconsisting in phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl,or heteroaromatics selected from the group comprising pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH₂)_(n)CH₃; thesubscript n is an integer independently selected from 0 to 15; Z is H,NO₂, OH, NR10R11 where R10 and R11 are each independently selected fromthe group consisting in H, (CH₂)_(n)CH₃, NHCOR12 where R12 is(CH₂)_(n)CH₃, aromatic and heteroaromatics selected form the groupcomprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH₂)_(n)CH₃, aromaticand heteroaromatics selected from the group comprising phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, NHSO₂R14 withR14 is phenyl, 2-, 3- or 4, substituted phenyl, naphthyl,heteroaromatics selected from the group comprising pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH₂)_(n)CH₃; thesubscript n is an integer independently selected from 0 to 15;
 6. The6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I, or one of itsderivatives having Notch signalling pathway inhibition properties, ofclaim 5 wherein the derivative having Notch signalling pathwayinhibition properties is selected from the group consisting in


7. A pharmaceutical composition comprising6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I,

or one of its derivatives having Notch signalling pathway inhibitionproperties, or pharmaceutically acceptable salts, solvates, tautomers,isomers thereof, and a pharmaceutically acceptable carrier.
 8. Thepharmaceutical composition of claim 7 wherein the derivative havingNotch signalling pathway inhibition properties is selected from thegroups comprising

wherein m is an integer selected from 1 to 4; W is selected from H andhalogens; the halogen is selected from F—, Cl—, Br— or I—; R1, R2, R3,R4 are each independently selected from the group consisting in H,phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl,furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl,tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n is an integerindependently selected from 1 to 15; X is O, S, CR5R6, NR7, NHCOR8, orNHSO2R9; where R5, R6, R7, R8, R9 are each independently selected fromthe group consisting in selected from the group consisting in H, phenyl,2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, or(CH2)_(n)CH3, the subscript n is an integer independently selected from1 to 15; Y is N or CH; Z is H, NO₂, OH, NR10R11 where R10 and R11 areeach independently selected from the group consisting in H and(CH₂)_(n)CH₃, NHCOR12 where R12 is selected from the group consisting of(CH₂)_(n)CH₃, aromatic and heteroaromatics such as phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, COOR13 whereR13 is selected from the group consisting of H, (CH₂)_(n)CH₃, aromaticand heteroaromatics such as phenyl, naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, NHSO₂R14 where R14 is selectedfrom the group consisting of phenyl, 2-, 3- or 4, substituted phenyl,naphthyl, heteroaromatics such as pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is aninteger independently selected from 1 to 15;

wherein m is an integer selected from 1 to 4; W is selected from H andhalogens; the halogen is selected from F—, Cl—, Br— or I—; R4, R15 areeach independently selected from the group consisting in H, phenyl, 2-,3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,(CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n is an integerindependently selected from 1 to 15; X is O, S, CR5R6, NR7, NHCOR8 orNHSO₂R9; R5, R6 and R7 are each independently selected from the groupconsisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃; R8 and R9 are eachindependently selected from the group consisting in phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected fromthe group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is an integerindependently selected from 1 to 15; Y is N or CH; Z is H, NO₂, OH,NR10R11 where R¹⁰ and R¹¹ are each independently selected from the groupconsisting in H, (CH₂)_(n)CH₃, NHCOR¹² where R12 is (CH₂)_(n)CH₃,aromatic and heteroaromatics selected from the group comprising phenyl,naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,COOR13 where R13 is H, (CH₂)_(n)CH₃, aromatic and heteroaromaticsselected from the group comprising phenyl, naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, NHSO₂R14 where R14 is phenyl, 2-,3- or 4, substituted phenyl, naphthyl, heteroaromatics selected from thegroup comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃, the subscript n is an integerindependently selected from 1 to 15;

wherein m is an integer selected from 1 to 4; W is selected from H andhalogens; the halogen is selected from F—, Cl—, Br— or I—; R4 is H,phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl,furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl,tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n is an integerindependently selected from 1 to 15; X is O, S, CR5R6, NR7, NHCOR8 orNHSO₂R9; R5, R6 and R7 are each independently selected from the groupconsisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃; R8 and R9 are eachindependently selected from the group consisting in phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected fromthe group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is an integerindependently selected from 1 to 15; Y is N or CH; Z is H, NO₂, OH,NR10R11 where R¹⁰ and R¹¹ are each independently selected from the groupconsisting in H, (CH₂)_(n)CH₃, NHCOR¹² where R12 is (CH₂)_(n)CH₃,aromatic and heteroaromatics selected from the group comprising phenyl,naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,COOR13 where R13 is H, (CH₂)_(n)CH₃, aromatic and heteroaromaticsselected from the group comprising phenyl, naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, NHSO₂R14 where R14 is phenyl, 2-,3- or 4, substituted phenyl, naphthyl, heteroaromatics selected from thegroup comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃, the subscript n is an integerindependently selected from 1 to 15;

wherein m is an integer selected from 1 to 4; W is selected from H andhalogens; the halogen is selected from F—, Cl—, Br— or I—; R1, R2, R3,R4 are each independently selected from the group consisting in H,phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl,furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl,tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n is an integerindependently selected from 1 to 15; X is O, S, CR5R6, NR7, NHCOR8 orNHSO₂R9; R5, R6 and R7 are each independently selected from the groupconsisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃; R8 and R9 are eachindependently selected from the group consisting in phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected fromthe group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is an integerindependently selected from 1 to 15; Z is H, NO₂, OH, NR10R11 where R10and R11 are each independently selected from the group consisting in H,(CH₂)_(n)CH₃, NHCOR12 where R12 is (CH₂)_(n)CH₃, aromatic andheteroaromatics selected form the group comprising phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, COOR13 whereR13 is H, (CH₂)_(n)CH₃, aromatic and heteroaromatics selected from thegroup comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4,substituted phenyl, naphthyl, heteroaromatics selected from the groupcomprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, (CH₂)_(n)CH₃; the subscript n is an integer independentlyselected from 1 to 15;

wherein m is an integer selected from 1 to 4; W is selected from H andhalogens; the halogen is selected from F—, Cl—, Br— or I—; R4, R15 areeach independently selected from the group consisting in H, phenyl, 2-,3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,(CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n is an integerindependently selected from 0 to 15; X is O, S, CR5R6, NR7, NHCOR8 orNHSO₂R9; R5, R6 and R7 are each independently selected from the groupconsisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃; R8 and R9 are eachindependently selected from the group consisting in phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected fromthe group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is an integerindependently selected from 0 to 15; Z is H, NO₂, OH, NR10R11 where R10and R11 are each independently selected from the group consisting in H,(CH₂)_(n)CH₃, NHCOR12 where R12 is (CH₂)_(n)CH₃, aromatic andheteroaromatics selected form the group comprising phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, COOR13 whereR13 is H, (CH₂)_(n)CH₃, aromatic and heteroaromatics selected from thegroup comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4,substituted phenyl, naphthyl, heteroaromatics selected from the groupcomprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, (CH₂)_(n)CH₃; the subscript n is an integer independentlyselected from 0 to 15;

wherein m is an integer selected from 1 to 3; W is selected from H andhalogens; the halogen is selected from F—, Cl—, Br— or I—; R4 is H,phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl,furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl,tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; the subscript n is an integerindependently selected from 0 to 15; X is O, S, CR5R6, NR7, NHCOR8 orNHSO₂R9; R5, R6 and R7 are each independently selected from the groupconsisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃; R8 and R9 are eachindependently selected from the group consisting in phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected fromthe group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is an integerindependently selected from 0 to 15; Z is H, NO₂, OH, NR10R11 where R10and R11 are each independently selected from the group consisting in H,(CH₂)_(n)CH₃, NHCOR12 where R12 is (CH₂)_(n)CH₃, aromatic andheteroaromatics selected form the group comprising phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, COOR13 whereR13 is H, (CH₂)_(n)CH₃, aromatic and heteroaromatics selected from thegroup comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4,substituted phenyl, naphthyl, heteroaromatics selected from the groupcomprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, (CH₂)_(n)CH₃; the subscript n is an integer independentlyselected from 0 to 15;

wherein the heteroaromatic is an aminopyrrole, aminofurane,aminothiofurane, or a pyrimidine; R1, R2, R3, R4 are each independentlyselected from the group consisting in H, phenyl, 2-, 3- or 4-substitutedphenyl, 2- or 3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl;the subscript n is an integer independently selected from 0 to 15; X isO, S, CR5R6, NR7, NHCOR8, or NHSO2R9; where R5, R6, R7, R8, R9 are eachindependently selected from the group consisting in H, phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, or (CH2)_(n)CH3,the subscript n is an integer independently selected from 0 to 15; Z isH, NO₂, OH, NR10R11 where R10 and R11 are each independently selectedfrom the group consisting in H, (CH₂)_(n)CH₃, NHCOR12 where R12 is(CH₂)_(n)CH₃, aromatic and heteroaromatics selected form the groupcomprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH₂)_(n)CH₃, aromaticand heteroaromatics selected from the group comprising phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, NHSO₂R14 withR14 is phenyl, 2-, 3- or 4, substituted phenyl, naphthyl,heteroaromatics selected from the group comprising pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH₂)_(n)CH₃; thesubscript n is an integer independently selected from 0 to 15;

wherein the subscript n is an integer independently selected from 1 to15; the heteroaromatic is an aminopyrrole, aminofurane, aminothiofurane,or a pyrimidine; R4, R15 are each independently selected from the groupconsisting in H, phenyl, 2-, 3- or 4-substituted phenyl, 2- or3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃ alkenyl, alkynyl; thesubscript n is an integer independently selected from 0 to 15; X is O,S, CR5R6, NR7, NHCOR8 or NHSO₂R9; R5, R6 and R7 are each independentlyselected from the group consisting in H, phenyl, 2-, 3- or 4-substitutedphenyl, 2- or 3-naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃; R8 and R9 areeach independently selected from the group consisting in phenyl, 2-, 3-or 4-substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selectedfrom the group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl,imidazolyl, benzyl, (CH₂)_(n)CH₃; the subscript n is an integerindependently selected from 0 to 15; Z is H, NO₂, OH, NR10R11 where R10and R11 are each independently selected from the group consisting in H,(CH₂)_(n)CH₃, NHCOR12 where R12 is (CH₂)_(n)CH₃, aromatic andheteroaromatics selected form the group comprising phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, COOR13 whereR13 is H, (CH₂)_(n)CH₃, aromatic and heteroaromatics selected from thegroup comprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, NHSO₂R14 with R14 is phenyl, 2-, 3- or 4,substituted phenyl, naphthyl, heteroaromatics selected from the groupcomprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl,benzyl, (CH₂)_(n)CH₃; the subscript n is an integer independentlyselected from 0 to 15;

wherein the heteroaromatic is an aminopyrrole, aminofurane,aminothiofurane, or a pyrimidine; R4 is H, phenyl, 2-, 3- or4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, (CH₂)_(n)CH₃alkenyl, alkynyl; the subscript n is an integer independently selectedfrom 0 to 15; X is O, S, CR5R6, NR7, NHCOR8 or NHSO₂R9; R5, R6 and R7are each independently selected from the group consisting in H, phenyl,2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl,(CH₂)_(n)CH₃; R8 and R9 are each independently selected from the groupconsisting in phenyl, 2-, 3- or 4-substituted phenyl, 2- or 3-naphthyl,or heteroaromatics selected from the group comprising pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH₂)_(n)CH₃; thesubscript n is an integer independently selected from 0 to 15; Z is H,NO₂, OH, NR10R11 where R10 and R11 are each independently selected fromthe group consisting in H, (CH₂)_(n)CH₃, NHCOR12 where R12 is(CH₂)_(n)CH₃, aromatic and heteroaromatics selected form the groupcomprising phenyl, naphthyl, pyrrolyl, furanyl, thiofuranyl,pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH₂)_(n)CH₃, aromaticand heteroaromatics selected from the group comprising phenyl, naphthyl,pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, NHSO₂R14 withR14 is phenyl, 2-, 3- or 4, substituted phenyl, naphthyl,heteroaromatics selected from the group comprising pyrrolyl, furanyl,thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH₂)_(n)CH₃; thesubscript n is an integer independently selected from 0 to 15;
 9. Thepharmaceutical composition of claim 8 wherein the derivative is selectedfrom the groups consisting in


10. A kit comprising one or more doses of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), or one of its derivativeshaving Notch signalling pathway inhibition properties, for use in amethod for treatment and/or prevention of cancer, optionally withreagents and/or instructions for use.
 11. The kit of claim 10, furthercomprising one or more doses of a chemotherapeutic agent.
 12. Use of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I

or one of its derivatives having Notch signalling pathway inhibitionproperties, for inhibiting in vitro the Notch signalling pathway incells.
 13. The use of claim 12 wherein the cells are cancer cells. 14.Use of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I

or one of its derivatives having Notch signalling pathway inhibitionproperties, for inhibiting in vivo the Notch signalling pathway incells.
 15. The use of claim 14 wherein the cells are cancer cells.
 16. Amethod of treating a subject for Notch dependent cancer, comprising i)determining in cancer cells obtained from a biological sample of saidsubject whether the cancer is Notch signalling pathway dependent, ii)and treating said subject based upon whether the cancer is Notchdependent cancer by administering a therapeutically effective amount of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I or one of itsderivatives having Notch signalling pathway inhibition properties, or apharmaceutical composition of any of claims 7 to
 9. 17. The method oftreating of claim 16 wherein the Notch signalling pathway dependency incancer cells is determined by an in vitro γ-secretase complex activityassay.
 18. The method of treating of claim 16 or 17 further comprisingadministering at least one conventional cancer treatment.
 19. The methodof treating of claim 18 wherein the conventional cancer treatment isadministered before, simultaneously or after the administration of thetherapeutically effective amount of6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3) of Formula I or one of itsderivatives having Notch signalling pathway inhibition properties, orthe pharmaceutical composition of any of claims 7 to
 9. 20. The methodof treating of claim 18 wherein the conventional cancer treatmentconsists in radiotherapy and/or chemotherapy.
 21. A method of treatmentof a disease associated with an up-regulated Notch signalling pathwayactivity, comprising administrating the6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), a derivative thereof, orthe pharmaceutical composition of claims 7 to 9 to a subject in needthereof.